doi:10.1152/ajpgi.00257.2009 298:63-73, 2010. First published Sep 17, 2009;Am J Physiol Gastrointest Liver Physiol

Yokosuka, Stephen J. Pandol and Anna S. Gukovskaya Damoiseaux, Moussa Yazbec, Ravinder Abrol, William A. Goddard, III, Osamu Izumi Ohno, Guido Eibl, Irina Odinokova, Mouad Edderkaoui, Robert D.

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Rottlerin stimulates apoptosis in pancreatic cancer cells through interactionswith proteins of the Bcl-2 family

Izumi Ohno,1,2,7 Guido Eibl,3 Irina Odinokova,1,2,4 Mouad Edderkaoui,1,2 Robert D. Damoiseaux,5

Moussa Yazbec,1,2 Ravinder Abrol,6 William A. Goddard, III,6 Osamu Yokosuka,7 Stephen J. Pandol,1,2

and Anna S. Gukovskaya1,2

1Veterans Affairs Greater Los Angeles Healthcare System, Departments of 2Medicine and 3Surgery, David Geffen Schoolof Medicine, and 5Molecular Shared Screening Resources, University of California, Los Angeles, California; 4Instituteof Theoretical and Experimental Biophysics, Pushchino, Russia; 6Materials and Process Simulation Center, BeckmanInstitute, California Institute of Technology, Pasadena, California; 7Department of Medicine and Clinical Oncology,Graduate School of Medicine, Chiba University, Chiba, Japan

Submitted 29 June 2009; accepted in final form 10 September 2009

Ohno I, Eibl G, Odinokova I, Edderkaoui M, Damoiseaux RD,Yazbec M, Abrol R, Goddard WA 3rd, Yokosuka O, Pandol SJ,Gukovskaya AS. Rottlerin stimulates apoptosis in pancreatic cancercells through interactions with proteins of the Bcl-2 family. Am JPhysiol Gastrointest Liver Physiol 298: G63–G73, 2010. First pub-lished September 17, 2009; doi:10.1152/ajpgi.00257.2009.—Rottlerinis a polyphenolic compound derived from Mallotus philipinensis. Inthe present study, we show that rottlerin decreased tumor size andstimulated apoptosis in an orthotopic model of pancreatic cancer withno effect on normal tissues in vivo. Rottlerin also induced apoptosisin pancreatic cancer (PaCa) cell lines by interacting with mitochondriaand stimulating cytochrome c release. Immunoprecipitation resultsindicated that rottlerin disrupts complexes of prosurvival Bcl-xL withBim and Puma. Furthermore, siRNA knockdown showed that Bimand Puma are necessary for rottlerin to stimulate apoptosis. We alsoshowed that rottlerin and Bcl-2 and Bcl-xL inhibitor BH3I-2� stimu-late apoptosis through a common mechanism. They both directlyinteract with mitochondria, causing increased cytochrome c releaseand mitochondrial depolarization, and both decrease sequestration ofBH3-only proteins by Bcl-xL. However, the effects of rottlerin andBH3I-2� on the complex formation between Bcl-xL and BH3-onlyproteins are different. BH3I-2� disrupts complexes of Bcl-xL with Badbut not with Bim or Puma, whereas rottlerin had no effect on theBcl-xL interaction with Bad. Also BH3I-2�, but not rottlerin, requiredBad to stimulate apoptosis. In conclusion, our results demonstrate thatrottlerin has a potent proapoptotic and antitumor activity in pancreaticcancer, which is mediated by disrupting the interaction betweenprosurvival Bcl-2 proteins and proapoptotic BH3-only proteins. Thusrottlerin represents a promising novel agent for pancreatic cancertreatment.

phytochemical; polyphone; mitochondria; proapoptotic protein

PANCREATIC ADENOCARCINOMA is an aggressive malignancy andis resistant to chemo- and radiotherapy (7, 38). One mechanismmediating pancreatic cancer aggressiveness and unresponsive-ness to treatment is its resistance to apoptosis. Finding ap-proaches to stimulate the death-signaling mechanisms is criti-cal for the development of effective therapeutic strategies inpancreatic cancer.

Rottlerin is a polyphenolic compound derived from Mallotusphilipinensis (Euphorbiaceae) (15). Rottlerin is widely used as

an inhibitor of the �-isoform of protein kinase C (15). Anothercellular target of rottlerin is mitochondria. Rottlerin was shownto cause uncoupling of mitochondrial respiration from oxida-tive phosphorylation and a collapse of mitochondrial mem-brane potential (��m) in several cell types (8). Recently,rottlerin was shown to stimulate apoptosis in various cancercells, including colon (40) and lung (6) cancer cells, chronicleukemia, and multiple myeloma cells (31, 34). The mecha-nisms whereby rottlerin stimulates apoptosis have not yet beenelucidated.

The key signaling event in apoptosis pathway is the releaseof mitochondrial cytochrome c into the cytosol, which isusually accompanied by a decrease in ��m. Once in thecytosol, cytochrome c activates caspases, leading to apoptosis.Proteins of the Bcl-2 family are major regulators of cyto-chrome c release. On the basis of their function and structure,Bcl-2 proteins can be divided into three groups, namely pro-apoptotic proteins containing three homologous BH domains(BH1-BH3), proapoptotic proteins containing BH3 domainonly, and prosurvival proteins containing four BH domains(BH1-BH4). Proapoptotic multidomain Bax and Bak formchannels in the outer mitochondrial membrane through whichmitochondrial cytochrome c is released into the cytosol (25,29). BH3-only proteins, such as Bad, Bim, and Puma, activateBax/Bak channels (1, 24, 46). Oppositely, prosurvival Bcl-2proteins, such as Bcl-xL and Bcl-2, bind to and sequester Bad,Bim, and Puma, preventing them from activating Bax/Bakchannels (1, 24, 28, 46).

During the past several years, small-molecule pharmacolog-ical inhibitors of prosurvival Bcl-2/Bcl-xL have been devel-oped and shown to stimulate apoptosis in cancer cells, dem-onstrating prosurvival Bcl-2 proteins as promising therapeutictargets in cancer treatment. At the same time, there is agrowing interest in testing the therapeutic potential and study-ing the mechanisms of action of natural phytochemical com-pounds as anticancer drugs (3, 21, 27).

The present study shows that rottlerin inhibits tumor growthin orthotopic models of pancreatic cancer and stimulates apop-tosis both in vivo in the pancreatic tumor and in vitro inpancreatic cancer (PaCa) cells. BH3-only proteins Bim andPuma are required for rottlerin to stimulate apoptosis. Rottlerinprevents sequestration of Bim and Puma by Bcl-xL, resultingin cytochrome c release, loss of ��m, and PaCa cell death.Rottlerin and the Bcl-2/Bcl-xL inhibitor, BH3I-2�, activate acommon signaling pathway in PaCa cells by preventing se-

Address for reprint requests and other correspondence: A. S. Gukovskaya,UCLA/VA Greater Los Angeles Healthcare System, West Los Angeles VAHealthcare Ctr., 11301 Wilshire Blvd, Bldg. 258, Rm. 340, Los Angeles, CA90073 (e-mail: agukovsk@ucla.edu)

Am J Physiol Gastrointest Liver Physiol 298: G63–G73, 2010.First published September 17, 2009; doi:10.1152/ajpgi.00257.2009.

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questration of BH3-only proteins by Bcl-xL, thus stimulatingapoptosis.

MATERIALS AND METHODS

Materials. Antibodies against caspase-3 caspase-9, PKC-�, and Bcl-2were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Bcl-xL, Puma, Bim, and Bad, total Akt, and phospho473S-Akt antibodieswere from Cell Signaling Technology (Denver, MA); anti-cytochrome cantibody was from BD Pharmingen (San Diego, CA); anti-cytochrome coxidase subunit IV antibody was from Molecular Probes (Carlsbad, CA);rottlerin was from Sigma (St. Louis, MO); BH3I-2� and PKC-� peptidesubstrate were from Calbiochem (San Diego, CA). PKC assay kit waspurchased from Upstate Biotechnology (Lake Placid, NY). All otherreagents were purchased from Sigma. PKC inhibitors GF-109203X andRo-32-0432 were from Calbiochem. A specific PKC-� translocationinhibitor (�V1-1: S-F-N-S-Y-E-L-G-S-L) was synthesized as we de-scribed previously (35).

Pancreatic cancer animal models. Animal studies were approvedby the Chancellor’s Animal Research Committee of the University ofCalifornia, Los Angeles in accordance with the NIH Guide for theCare and Use of Laboratory Animals. One model used was thesubcutaneous model. Pancreatic cancer cells (MIA PaCa-2, 2 � 106)were subcutaneously injected into the flanks of nude mice. After thetumor reached a size of �2 � 2 mm, animals were randomly allocatedto receive either control vehicle (n � 6) or rottlerin (0.5 mg/kg, n �6). Drugs were prepared fresh each day and injected intraperitoneallyin a total volume of 50 �l for 2 wk. The other model used was theorthotopic model. Pancreatic tumors grown subcutaneously wereharvested, and tumor pieces (1 mm3) were transplanted into the tail ofthe pancreas of recipient nude mice (20). Animals were randomizedand treated with either daily intraperitoneal injections of rottlerin (0.5mg/kg, n � 8) or control vehicle (n � 8) for 4 wk. At euthanasia,pancreatic tumors, liver, spleen, lungs, and kidney were harvested.Tumor volume was assessed as described earlier (12). For furtheranalyses, the tumor was either fixed in formalin and embedded inparaffin or frozen in 2-methyl-butane/dry ice and embedded in opti-mal cutting temperature compound.

In situ TUNEL assay. The percentage of apoptotic cells in tissuesections was determined as described previously (16). Briefly, cryo-stat sections were fixed in 4% paraformaldehyde, and in situ terminaldeoxynucleotidyl transferase-mediated nick end labeling (TUNEL)assay was done as described by the manufacturer (Roche MolecularBiochemicals, Manheim, Germany). The number of apoptotic cellswas determined in relation to the total number of cells.

Cell culture. Human pancreatic adenocarcinoma cell lines, thepoorly differentiated MIA PaCa-2, and moderately differentiatedPANC-1 were obtained from the American Type Culture Collection(Manassas, VA). MIA PaCa-2 and PANC-1 cells were grown in 1:1DMEM-F-12 medium from GIBCO Invitrogen (Grand Island, NY)supplemented with 15% FBS, 4 mM L-glutamine, and antibiotic/antimycotic solution from Omega Scientific (Tarzana, CA). Cellswere incubated at 37°C in a humidified atmosphere containing 5%CO2 and were used between passages 4 and 12. For analyses, cellswere plated at a density of 5 � 105/ml in either 100-mm culture dishesor 150 cm2, and cells were cultured for up to 48 h.

Transfection. Puma, Bim, Bad, and negative control siRNAs wereobtained from Dharmacon (Lafayette, CO). PaCa cells were tran-siently transfected using Nucleofector system from Amaxa Biosys-tems (Cologne, Germany). The 2 � 106 cells were resuspended incorresponding Nucleofector solution. siRNA and cells were mixed inthe electroporation Amaxa cuvette, and transfection was performedwith the electrical setting T20. After nucleofection the cells wereimmediately transferred into prewarmed medium and cultured at37°C, 5% CO2.

Measurement of apoptosis in cells. Internucleosomal DNA frag-mentation was measured by using Cell Death Detection ELISAPlus kit

(Roche Molecular Biochemicals) according to the manufacturer’sinstructions. Phosphatidylserine externalization was analyzed with theannexin-V (AnV)-FLUOS Staining Kit from Roche Biochemicals (Indi-anapolis, IN) as we described before (9, 41, 42). Caspase-3, -9, and -8activities were measured as we described previously (9, 41, 42) using afluorogenic assay with the substrates specific for caspase-3 [Ac-DEVDamino-4-methylcoumarin (AMC)], caspase-9 (Ac-LEHD-AMC), andcaspase-8 (Ac-IETD-AMC). The data were expressed as moles ofAMC/mg of protein/min and normalized to the control.

Cell fractionation for the measurement of cytochrome c release.Cells were resuspended in a lysis buffer (250 mM sucrose, 20 mMHEPES, 10 mM KCl, 1 mM Na-EGTA, 1 mM Na-EDTA, 2 mMMgCl2, pH 7.0); disrupted by 80 strokes in a Dounce homogenizer, andcentrifuged at 1,000 g to pellet nuclei and cell debris. Supernatants werefurther centrifuged at 13,000 g for 30 min, and the cytosolic fractions(supernatants) and the pellets (membrane fractions enriched with mito-chondria) were collected. To validate the quality of cytosolic and mito-chondrial separation, both fractions were assessed by immunoblotting forthe mitochondrial marker cytochrome c oxidase subunit IV.

Total cell lysates. Cells were resuspended in RIPA phosphorylationbuffer (50 mM NaCl, 50 mM Tris �HCl pH 7.2, 1% deoxycholic acid,1% Triton X-100, 0.1% SDS, 10 mM Na2HPO4 NaH2PO4 pH 7.0,100 mM NaF, 2 mM Na3VO4, 20% glycerol, 80 �M glycerophos-phate, 1 mM PMSF, 5 �g/ml of pepstatin, leupeptin, chymostatin,antipain, and aprotinin), sonicated, and centrifuged for 15 min at15,000 g at 4°C.

Immunoprecipitation. Cells were collected, washed twice in abuffer containing 20 mM Tris (pH 7.5) and 10 mM DTT, thenresuspended in a lysis buffer (50 mM Tris �HCl, 150 mM NaCl, 2 mMEGTA, 10 �g/ml each leupeptin and aprotinin, 1 mM PMSF, 1%NP-40), and sonicated for 30 s. Lysates were clarified by centrifuga-tion, and 500 �g of protein was subjected to overnight immunopre-cipitation with either Bcl-xL or Bcl-2 antibody at 4°C using Catch andRelease Reversible Immunoprecipitation System from Millipore (Bil-lerica, MA).

Western blot analysis. Proteins from total cell lysates or immuno-precipitates were separated by SDS-PAGE and electrophoreticallytransferred to nitrocellulose membranes. Nonspecific binding wasblocked with 5% bovine serum albumin or nonfat dry milk inTris-buffered saline (4 mM Tris base, 100 mM NaCl, pH 7.5)containing 0.05% Tween 20 for 1 h. Membranes were incubated withprimary antibody for overnight at 4°C and then for 1 h with peroxi-dase-conjugated secondary antibody. The blots were developed withSupersignal Chemiluminescent Substrate from Pierce (Rockford, IL).

Measurements of ��m in intact PaCa cells. Changes in ��m weredetected with the potential-sensitive probe MitoTracker Red (CMX-Ros) from Invitrogen (Carlsbad, CA) as we described previously (41).During the last 30 min of the incubation period, cells were loaded with10 nM CMX-Ros for 30 min at 37°C in the dark, washed twice withphosphate-buffered saline, and analyzed by flow cytometry using theFACScan with FL-3 detector. To completely dissipate ��m, cellswere treated with the uncoupling agent CCCP (50 �M) for 1 h beforeCMX-Ros staining.

Measurements of ��m and cytochrome c release in PaCa cellspermeabilized with digitonin. For permeabilization, cells were resus-pended in the medium that mimics ionic composition of cytosol. Itcontained 250 mM sucrose, 22 mM KCl, 11 mM triethanolamine, 3mM MgCl2, 5 mM KH2PO4 (pH 7.0), 0.5 mM EGTA, and 10 mMsuccinate incubated for 3 min with 1.25 � 103% digitonin asdescribed (24, 26). Efficiency of permeabilization was confirmed bypositive Trypan blue staining in greater than 90% of the cells.Changes in ��m were measured with a ��m-sensitive tetraphenylphosphonium (TPP)-selective electrode (23). An increase in ��mcauses TPP uptake by mitochondria and, correspondingly, a de-crease in external TPP measured by the electrode. Cytochrome clevels in soluble and membrane fractions were measured with West-

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ern blot. Cytochrome c release and ��m were measured in the samepreparations of permeabilized cells.

Fluorescence polarization assay. Complex formation between afluorescein-labeled peptide [NLWAAQRYGRELRRMSDK(fluores-cein)FVD] of BH3 domain of Bad (synthesized by CURE Researchcenter Peptide CORE) and the recombinant Bcl-xL fragment lackingthe flexible loop and the carboxyterminal hydrophobic region (Cal-biochem) was performed as described in Ref. 48. Briefly, Bcl-xL wasmixed with 15 nM fluorescently labeled Bad in a 96-well plate withand without rottlerin, BH3I-2�, or vehicle in a final volume of 50 �l,and fluorescence polarization was measured using Analyst 96-wellplate reader (LJL; Molecular Devices, Sunnyvale, CA).

Measurements of PKC-� activity. Measurements of PKC-� activitywere performed as we described in Ref. 35. Briefly, cells werehomogenized in ice-cold homogenization buffer containing (in mM)130 NaCl, 50 Tris �HCl (pH 7.5), 5 EGTA, 5 EDTA, 1.5 MgCl2, 10NaF, 1 Na3VO4, 10 Na4P2O7, 1 PMSF, and 10% (vol/vol) glycerolplus 5 �g/ml each of pepstatin, leupeptin, chymostatin, antipain, andaprotinin, sonicated five times for 10 s on ice, and incubated for 45min at 4°C. PKC-� was immunoprecipitated using specific antibodyagainst PKC-� isoform (1:100 dilution). The beads were washed andresuspended in the kinase buffer [(in mM) 20 MOPS (pH 7.2), 25�-glycerophosphate, 5 EGTA, 1 Na3VO4, and 1 DTT]. The kinaseassay was performed by using the PKC assay kit (Upstate Biotech-nology).

Statistical analysis. For statistical analysis, the results were from atleast three independent experiments. Differences between two groupswere analyzed using Student’s t-test. P � 0.05 was consideredstatistically significant.

RESULTS

Rottlerin inhibited tumor growth and stimulated apoptosis inpancreatic cancer animal models. To determine the effects ofrottlerin on the growth of xenografted pancreatic cancers weused a subcutaneous and orthotopic pancreatic cancer model innude mice as described before (11, 12, 20). Mice receivedeither 0.5 mg/kg of rottlerin or vehicle (DMSO) daily for 2(subcutaneous model) or 4 wk (orthotopic model). There wasno difference in body weight throughout the study periodbetween both experimental groups (not shown). In both mod-els, rottlerin significantly inhibited tumor growth by 60–70%(Fig. 1, A and B). The decrease in tumor growth was associatedwith an increase in apoptotic cell death in PaCa cells. In theorthotopic model, treatment of mice with rottlerin increasedapoptosis in PaCa cells fourfold compared with control animals(Fig. 1C), suggesting that rottlerin inhibits pancreatic cancergrowth by induction of apoptosis. Importantly, normal extra-tumoral tissues, e.g., liver and kidney, were histologicallynormal; there was no increase in apoptosis or necrosis in theseorgans (not shown).

Rottlerin stimulated PaCa cell death. Rottlerin at micromo-lar concentrations dose dependently stimulated apoptosis inPaCa cells (Fig. 2). Increase in DNA fragmentation in bothMIA PaCa-2 and PANC-1 cells was already significant at 1.3�M and reached maximum at 2.5 �M rottlerin (Fig. 2A). Ofnote, application of 2.5 �M rottlerin in vitro is approximatelyequivalent to the in vivo application of 0.5 mg/kg. Stimulationof apoptosis by rottlerin increased with the time and reachedplateau at 24-h culturing with rottlerin.

To assess the differential effects of rottlerin on apoptosis andnecrosis, we stained MIA PaCa-2 cells with AnV and pro-pidium iodide (PI). As we showed before (41), AnV/PI

group represents cells with early apoptosis; cells stained with

PI only (AnV/PI) are early necrotic ones, whereas AnV/PI group includes cells with primary necrosis and also cellswith apoptosis associated with secondary necrosis (28a, 41).Rottlerin increased the number of cells in each group up to

Fig. 1. Rottlerin inhibited pancreatic cancer growth in vivo. The effects ofrottlerin on pancreatic cancer growth were measured in a subcutaneous (A) andorthotopic (B) xenograft model in nude mice. Animals received either rottlerin(0.5 mg/kg) or vehicle by daily intraperitoneal injections. The tumor volumesof rottlerin- and vehicle- treated animals were determined after 2 wk in thesubcutaneous (A) and after 4 wk in the orthotopic model (B). C: percentage ofapoptotic cells in tissues sections was determined in cryostat sections fixed in4% paraformaldehyde using terminal deoxynucleotidyl transferase-mediatednick end labeling assay. The number of apoptotic cells was determined inrelation to the total number of cells. The values in A–C represent means SE(at least 5 animals in each group). *P � 0.05, **P � 0.02, ***P � 0.0005 vs.the same parameter in tumors from mice treated without rottlerin.

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twofold and as a result decreased the number of live (AnV/PI) cells (Fig. 2B). These data indicate that rottlerin stimu-lated not only apoptotic but also necrotic death of PaCa cells.

We compared the proapoptotic effects of rottlerin with thoseinduced by BH3I-2�, an inhibitor of Bcl-2 and Bcl-xL. BH3I-2�is a mimetic of BH3 domain; it binds to the hydrophobicpocket of Bcl-2 and Bcl-xl, preventing their interaction withBH3-only proteins (13, 18). BH3I-2’ stimulated apoptosis inboth MIA PaCa-2 and PANC-1 cells (Fig. 2C). The effects of25 �M of BH3I-2� and 2.5 �M of rottlerin on apoptosis werenonadditive, suggesting a common underlying mechanism(Fig. 2D).

Rottlerin stimulated mitochondrial pathway of apoptosis inPaCa cells. Rottlerin dose and time dependently stimulatedcytochrome c release from mitochondria into cytosol in bothMIA PaCa-2 and PANC-1 cells (Fig. 3, A and B). Cytochromec release increased with the concentration of rottlerin in the

range of 2.5 to 10 �M. In MIA PaCa-2 cells, the induction ofcytochrome c release was evident as early as at 15 min andcontinued to increase for up to 48 h (Fig. 3, A and B). BH3I-2�also dose dependently stimulated cytochrome c release in MIAPaCa cells (Fig. 3C).

We next examined the direct effect of rottlerin and BH3I-2�on mitochondria using MIA PaCa-2 cells permeabilized withdigitonin (Fig. 3D). When added to permeabilized cells bothrottlerin and BH3I-2� dose-dependently stimulated cytochromec release (Fig. 3D), suggesting that both these agents stimulatecytochrome c release by directly affecting mitochondria.

Cytochrome c release in apoptosis is usually associated withmitochondrial depolarization (14). Rottlerin dose dependentlydecreased ��m in MIA PaCa-2 and PANC-1 cells (Fig. 4A).Depolarization was first observed at 15 min of rottlerin treat-ment. Importantly, low concentrations of rottlerin (2.5 �M)transiently depolarized mitochondria, whereas higher concen-

Fig. 2. Rottlerin stimulated apoptosis in pancreatic cancer (PaCa) cells. MIA PaCa-2 and PANC-1 cells were cultured for indicated times (A) or for 48 h(B, C, and D) with indicated concentrations of rottlerin (A and B), BH3I-2� (C), combination of 2.5 �M rottlerin, and 25 �M BH3I-2� (D), or vehicle. Apoptosiswas determined by measuring DNA fragmentation (A, C, and D) and annexin-V/propidium iodide (AnV/PI) staining (B). A, C, and D: DNA fragmentation wasmeasured by Cell Death ELISA kit. The values were normalized to those in cells cultured without rottlerin. B: cells were stained with AnV and PI, analyzedby flow cytometry, and the percentages of cells in groups AnV/PI (early apoptosis), AnV/PI (early necrosis), AnV/PI (mixture of apoptosis associatedwith secondary necrosis and late necrosis), and AnV/PI (live cells) were measured. The values in represent means SE, n � 3.

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trations (5 and 10 �M) caused permanent loss of ��m (Fig.4A). The reason for transient depolarization remains to bedetermined. However, the differences in the time dependenciesof rottlerin-induced cytochrome c release and depolarizationsuggest that cytochrome c release and depolarization are me-diated through different mitochondrial permeability systems.BH3I-2� also dose dependently stimulated mitochondrial de-polarization of MIA PaCa cells (Fig. 4B). In contrast torottlerin, the effect of BH3I-2� on ��m was not transient atconcentrations tested.

Both rottlerin and BH3I-2� stimulated loss of ��m indigitonin-permeabilized MIA PaCa-2 cells (Fig. 4, C and D),indicating direct interaction of rottlerin and BH3I-2� withmitochondria.

Once in cytosol, cytochrome c activates the initiatorcaspase-9, followed by activation of effector caspase-3, result-ing in apoptosis (33). We found that rottlerin increased activity

of caspase-9 (LEHDase) as well as caspase-9 processing man-ifest by a decrease in procaspase-9 (47 kDa) and increase in35-kDa cleaved form (Fig. S1A; supplemental material for thisarticle is available online at the American Journal of Physiol-ogy Gastrointestinal and Liver Physiology website). Rottlerindose and time dependently stimulated caspase-3 (DEVDase)activity and its processing, which also manifests by a decreasein 32-kDa band of procaspase-3 and by a concomitant increasein 17-kDa band corresponding to active (i.e., cleaved)caspase-3 (Fig. S1, A and C). Of note, apoptosis induced byrottlerin in MIA PaCa-2 cells was prevented by pan-caspaseinhibitor z-VAD-fmk, indicating a necessary role of caspases(not shown). Importantly, rottlerin did not activate caspase-8(Fig. S1B), which acts upstream of mitochondria (33). Theseconfirm that it stimulates mitochondrial pathway of apoptosis.

The results of the Figs. 3, 4, and S1 suggest that rottlerin andBH3I-2� stimulate apoptosis through a common mechanism;

Fig. 3. Rottlerin stimulated cytochrome c(cyto c) release in PaCa cells. MIA PaCa-2cells (A and C) and PANC-1 cells (B) werecultured for indicated times with indicatedconcentrations of rottlerin (A and B),BH3I-2� (C), or vehicle. A–C: cytochrome clevels in cytosolic and membrane fractionswere measured with Western blot analysis.The blots were reprobed for actin and CoxIV to confirm equal protein loading in cyto-solic (actin) and membrane fractions(CoxIV). D: MIA PaCa-2 cells were perme-abilized for 3 min with 1.25 � 103% dig-itonin and then incubated with indicatedconcentrations of rottlerin (left) or BH3I-2�(right) for 15 min. Cytochrome c levels inthe medium were measured using Westernblot analysis. The blots were reprobed forCox IV to confirm no contamination of themedium with broken mitochondria and toconfirm equal amount of mitochondria in allconditions. Immunoblots are representativeof 3 independent experiments, which allgave the same results.

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they both directly interact with mitochondria, stimulating cy-tochrome c release and loss of ��m. This leads to caspase-9and -3 activation and apoptosis.

BH3I-2� but not rottlerin stimulated apoptosis through dis-rupting complexes of Bcl-xL with Bad. The pharmacologicalinhibitors of prosurvival of Bcl-xL and Bcl-2 induce apoptosisby preventing sequestration by prosurvival proteins of pro-apoptotic BH3-only proteins (18). Our immunoprecipitationexperiments showed that, in MIA PaCa-2 cells, the proapop-totic BH3-only protein Bad is in complex with Bcl-xL andBcl-2 (Fig. 5A). BH3I-2� markedly decreased the amount ofBad coimmunoprecipitated with Bcl-xL and Bcl-2 (Fig. 5, Aand B). Differently, rottlerin only slightly decreased theamount of Bad in complex with Bcl-xL (Fig. 5, A and B) andhad no effect on the interaction of Bad with Bcl-2 (Fig. 5A).

We further showed that Bad knockdown with siRNA greatlydecreased apoptosis induced by BH3I-2� but had no effect onapoptosis induced by rottlerin (Fig. 5, C and D), indicating thatproapoptotic effect of rottlerin does not involve Bad.

To compare the effects of rottlerin and BH3I-2� on theinteraction of Bcl-xL with Bad in cell-free system we exam-ined the effect of rottlerin on the binding of fluorescein-labeledpeptide of the BH3 domain of Bad protein to Bcl-xL usingfluorescence polarization assay (48). Figure S2 shows thatrecombinant Bcl-xL forms complex with the BH3 domain ofBad manifested by an increase in the fluorescence polarization.

BH3I-2� dose dependently disrupted complex formation be-tween Bcl-xL and BH3 domain of Bad, whereas rottlerin atconcentrations up to 200 �M was without effect. Thus theresults in MIA PaCa-2 cells, as well as the results in cell-freesystem, indicate that BH3I-2� but not rottlerin disrupts thecomplex of Bad with Bcl-xL and Bcl-2.

Rottlerin stimulated apoptosis through disrupting complexesof Bcl-xL with Bim and Puma. To determine whether theproapoptotic effect of rottlerin in PaCa cells is mediated throughPuma and Bim we measured the effect of rottlerin on the seques-tration of these BH3-only proteins by Bcl-xL (Fig. 6). Rottleringreatly decreased the amount of Puma and Bim coimmunopre-cipitated with Bcl-xL (Fig. 6, A and B). Differently, BH3I-2�had no effect on the interaction of Bcl-xL with Puma (Fig. 6A,B). Of note, neither rottlerin nor BH3I-2� affected the interac-tion of Bcl-2 with Puma or Bim (data not shown).

We further showed that Puma and Bim knockdown withsiRNA prevented stimulation of apoptosis by rottlerin in MIAPaCa-2 cells, indicating a critical role for Puma and Bim in theproapoptotic effects of rottlerin (Fig. 6, C and D). Of note,Puma and Bim knockdown with siRNA also prevented stimu-lation of apoptosis by rottlerin in PANC-1 cells (not illus-trated), suggesting that the mechanisms of proapoptotic effectsof rottlerin are the same in both MIA PaCa-2 and PANC-1cells.

Fig. 4. Rottlerin stimulated loss of ��m in PaCa-2 cells. MIA PaCa-2 (A and B) and PANC-1 (A) cells were cultured for indicated times (A) or 48 h (B) withindicated concentrations of rottlerin (A), BH3I-2� (B), or vehicle. A and B: mitochondrial membrane potential was measured in cells labeled with ��m-sensitivedye, MitoTracker Red (CMX-Ros) and analyzed with flow cytometry. The values in A and B were normalized to those at 0 time (A) or 48 h (B) in the absenceof rottlerin or BH3I-2�. The values represent means SE, n � 3. C and D: MIA PaCa-2 cells were permeabilized for 3 min with 1.25 � 103% digitonin andthen incubated with indicated concentrations of rottlerin (C), BH3I-2�(D), CCCP, or vehicle for indicated times. Changes in ��m were measured using��m-sensitive tetraphenyl phosphonium (TPP) electrode. Protonophore CCCP was applied to cancel membrane potential. The results are representative of 3independent experiments, which all gave the same results.

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Rottlerin-induced apoptosis was not mediated by the inhibi-tion of PKC-� or Akt. Apoptotic effects of rottlerin were oftenattributed to the inhibition of PKC-� (32, 36). Therefore, wenext examined the involvement of PKC-� in the effects ofrottlerin on apoptosis. We showed (Fig. 7A) that neither broadspectrum PKC inhibitor nor specific peptide inhibitor of PKC-�(35) stimulated apoptosis in MIA PaCa-2 cells. We alsoshowed that the inhibitors blocked PKC-� activity in MIAPaCa-2 cells (Fig. 7B and Ref. 46a). Pretreatment with PKCinhibitors also did not prevent apoptosis induced by rottlerin(Fig. 7C). Furthermore, rottlerin similarly stimulated apop-tosis in MIA PaCa-2 cells, which express PKC-�, and inPANC-1 cells, which do not express this isoform of PKC.

(17) As shown in several publications (33, 36), PKC-� couldinhibit apoptosis through activating prosurvival Akt kinase.However, rottlerin did not have any significant effect on Aktphosphorylation in PaCa cells (Fig. 7D). These data indicatethat PKC-� and Akt are not involved in proapoptotic effectsof rottlerin.

DISCUSSION

The results of this study show that rottlerin inhibited tumordevelopment and increased apoptosis in the orthotopic modelof pancreatic cancer. Rottlerin also stimulated apoptosis inPaCa cell lines.

Fig. 5. BH3I-2�, but not rottlerin, decreased the amount of Bad sequestered by Bcl-xL. Bad is required for apoptosis induced by BH3I-2� but not by rottlerin.A and B: MIA PaCa-2 cells were cultured for 1 h with 25 �M BH3I-2�, 2.5 �M of rottlerin, or vehicle. Bcl-xL and Bcl-2 were immunoprecipitated from totalcell lysates. A: immunoprecipitates (IP) were immunoblotted (IB) with antibody against Bcl-xL, Bcl-2, and Bad as indicated. B: intensities of the bands weredensitometrically quantified and expressed as the ratios of the intensities of Bad to that of Bcl-xL or Bcl-2. The ratios were normalized to those in cells culturedwithout BH3I-2� and rottlerin. The results are means SE of 2 experiments. C and D: MIA PaCa-2 cells were transfected with Bad siRNA or negative-controlsiRNA. C: transfection efficiency and lack of the effect of Bad knockdown on the levels of Puma and Bim were confirmed by immunoblot. D: transfected cellswere cultured for 24 h with 2.5 �M rottlerin (B), 25 �M BH3I-2�, or vehicle. DNA fragmentation was measured by Cell Death ELISA kit. The values werenormalized to those in cells cultured without rottlerin or BH3I-2�. Values are means SE (n � 3); *P � 0.05 compared with cells transfected with control siRNAand incubated without BH3I-2� and rottlerin.

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Several lines of evidence indicate that rottlerin inducedapoptosis in PaCa cells through interacting with Bcl-2 proteins.1) The proapoptotic effects of rottlerin were not mediated byPKC-� inhibition, as other inhibitors of PKC-� do not havesuch effect. 2) Rottlerin directly interacts with mitochondria,resulting in cytochrome c release and mitochondrial depolar-ization. 3) Rottlerin displaces Bim and Puma from complexeswith Bcl-xL. 4) Bim and Puma are necessary for rottlerin tocause apoptosis because their knockdown prevented inductionof apoptosis by rottlerin. Taken together, these findings providestrong support for our hypothesis that rottlerin acts to causeapoptosis through disrupting the ability of prosurvival Bcl-2proteins to sequester proapoptotic BH3-only proteins, Bim, andPuma.

The conclusion that proapoptotic effects of rottlerin aremediated through Bcl-2 proteins is further supported by thefindings that rottlerin has actions similar to a known inhibitorof Bcl-2/Bcl-xL, BH3I-2� (13, 18). BH3I-2� is a mimetic ofBH3 domain; it binds to the hydrophobic pocket of Bcl-2 andBcl-xL, preventing their interaction with BH3-only proteins(13, 18). The effects of BH3I-2� and rottlerin on apoptosis were

not additive. Rottlerin and BH3I-2� both directly affectedmitochondria, resulting in cytochrome c release and loss of��m. Rottlerin and BH3I-2� both disrupted complexes ofBcl-xL and BH3-only proteins. Proapoptotic effects of rottlerinand BH3I-2� are mediated by BH3-only proteins.

Importantly, we found that effects of rottlerin andBH3I-2� on the interaction of Bcl-xL with BH3-only pro-teins are not the same. Rottlerin prevented coimmunopre-cipitation of Bcl-xL with Bim and Puma, but it had littleeffect on the complex of Bcl-xL with Bad. Differently,BH3I-2� decreased the level of Bad, but not Bim and Puma,in the complex with Bcl-xL. The mechanisms underlyingsuch differences in the interaction of rottlerin and BH3I-2�with Bcl-xL remain to be determined.

Rottlerin stimulated not only cytochrome c release but alsoloss of ��m in PaCa cells. Data from other cell types showthat cytochrome c release and mitochondrial depolarization aremediated through different mitochondrial permeability systems(5). Cytochrome c is released through the Bax/Bak channels inthe outer mitochondrial membrane, whereas mitochondrialdepolarization is mediated by permeability transition pore

Fig. 6. Rottlerin decreased the amount of Puma and Bim sequestered by Bcl-xL. Puma and Bim are required for apoptosis induced by rottlerin. A and B: MIAPaCa-2 cells were cultured for 1 h with 2.5 �M of rottlerin, 25 �M BH3I-2�, or vehicle. Bcl-xL and Bcl-2 were immunoprecipitated from total cell lysates.A: immunoprecipitates were immunoblotted with antibody against Bcl-xL, Puma, Bim as indicated. B: intensities of the bands were densitometrically quantifiedand expressed as the ratios of the intensities of Puma or Bim to that of Bcl-xL. The ratios were normalized to those in cells cultured without rottlerin BH3I-2�and rottlerin. The results are means range of 2 experiments. C and D: MIA PaCa-2 cells were transfected with Puma or Bim siRNA or negative-control siRNA.C: transfection efficiency and lack of the effects of Puma or Bim knockdown on the Bad levels were confirmed by immunoblot. D: transfected cells were culturedfor 24 h with 2.5 �M rottlerin, 25 �M BH3I-2�, or vehicle. DNA fragmentation was measured by Cell Death ELISA kit. The values were normalized to thosein cells cultured without rottlerin or BH3I-2�. Values are means SE (n � 3).

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(mPTP), which involves proteins of both inner and outermembranes. Bcl-2 proteins were shown to be involved in theregulation of both mPTP and Bax/Bak channels (2, 4), whichexplains the effect of rottlerin on both cytochrome c releaseand ��m in PaCa cells.

In nontransformed cells, mitochondria are the major sourceof ATP production; therefore, mitochondrial depolarizationleads to ATP depletion and necrosis. ATP depletion alsoprevents caspase activation, leading to the inhibition of apop-tosis (10, 37, 47). Differently, in cancer cells, ATP productionis mostly mediated through glycolysis (43–45) and is relativelyindependent on ��m (19, 43, 44). In agreement with theseconsiderations, we found that, in PaCa cells (differently from

normal pancreatic acinar cells) (36, 39), rottlerin stimulatedboth apoptosis and necrosis.

Importantly, the results of in vivo experiments suggest thatnormal and cancer cells display different sensitivity to rottlerin.The doses of rottlerin we applied in vivo stimulated neitherapoptosis nor necrosis in normal pancreas and liver. Thesesame doses of rottlerin greatly potentiated apoptosis in pancre-atic tumor. Although the mechanisms underlying these differ-ences remain to be determined, the findings that low doses ofrottlerin to preferentially stimulate apoptosis in cancer cells areimportant for possible rottlerin application in cancer treatment.

In summary, our data showed that rottlerin has a potentproapoptotic and antitumor activity in pancreatic cancer. The

Fig. 7. Rottlerin-induced apoptosis is not mediated through PKC or Akt. MIA PaCa-2 cells were cultured for 48 h with indicated concentrations of specificPKC-� translocation inhibitor, broad-spectrum PKC inhibitors GF-109203X and R032–0432, rottlerin, or vehicle. A and C: apoptosis was determined bymeasuring DNA fragmentation using Cell Death ELISA kit. The values were normalized to those in cells cultured without inhibitors. The values are means SE, n � 3. B: changes in PKC-� kinase activities were measured in PKC-� immunoprecipitates by kinase assay using PKC-�-specific peptide substrate. Activitieswere normalized to the basal activity in the absence of the inhibitors. Values are means SE (n � 2). C: Akt phosphorylation, an indicator of Akt activation,was measured by immunoblots of Akt phosphorylated at 473S and total Akt. D: intensities of pAkt and total Akt were densitometrically quantified and expressedas the ratios of the intensities of phosphoAkt to that of total Akt. The values are means SE, n � 3.

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proapoptotic effect of rottlerin is mediated by the disruption ofthe complexes between prosurvival Bcl-2 proteins and BH3-only proteins Puma and Bim. Thus rottlerin represents apromising novel agent for pancreatic cancer treatment.

GRANTS

This study was supported by the Hirshberg Foundation for PancreaticCancer Research, the NIH grants CA119025 (to A. Gukovskaya.), NCCAMAT003960 (to S. Pandol), and the Department of Veterans Affairs MeritAward (to A. Gukovskaya).

DISCLOSURES

No conflicts of interest are declared by the author(s).

REFERENCES

1. Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer developmentand therapy. Oncogene 26: 1324–1337, 2007.

2. Bagci EZ, Vodovotz Y, Billiar TR, Ermentrout GB, Bahar I. Bistabil-ity in apoptosis: roles of bax, bcl-2, and mitochondrial permeabilitytransition pores. Biophys J 90: 1546–1559, 2006.

3. Bhattacharyya S, Mandal D, Saha B, Sen GS, Das T, Sa G. Curcuminprevents tumor-induced T cell apoptosis through Stat-5a-mediated Bcl-2induction. J Biol Chem 282: 15954–15964, 2007.

4. Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T,Korsmeyer SJ. BCL-2, BCL-X(L) sequester BH3 domain-only moleculespreventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell 8:705–711, 2001.

5. Chipuk JE, Bouchier-Hayes L, Green DR. Mitochondrial outer mem-brane permeabilization during apoptosis: the innocent bystander scenario.Cell Death Differ 13: 1396–1402, 2006.

6. Clark AS, West KA, Blumberg PM, Dennis PA. Altered protein kinaseC (PKC) isoforms in non-small cell lung cancer cells: PKCdelta promotescellular survival and chemotherapeutic resistance. Cancer Res 63: 780–786, 2003.

7. Crane CH, Ben-Josef E, Small W Jr. Chemotherapy for pancreaticcancer. N Engl J Med 350: 2713–2715; author reply 2713–2715, 2004.

8. Duchen MR. Roles of mitochondria in health and disease. Diabetes 53,Suppl 1: S96–S102, 2004.

9. Edderkaoui M, Hong P, Vaquero EC, Lee JK, Fischer L, Friess H,Buchler MW, Lerch MM, Pandol SJ, Gukovskaya AS. Extracellularmatrix stimulates reactive oxygen species production and increases pan-creatic cancer cell survival through 5-lipoxygenase and NADPH oxidase.Am J Physiol Gastrointest Liver Physiol 289: G1137–G1147, 2005.

10. Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levels determinecell death fate by apoptosis or necrosis. Cancer Res 57: 1835–1840, 1997.

11. Eibl G, Reber HA, Wente MN, Hines OJ. The selective cyclooxygen-ase-2 inhibitor nimesulide induces apoptosis in pancreatic cancer cellsindependent of COX-2. Pancreas 26: 33–41, 2003.

12. Eibl G, Takata Y, Boros LG, Liu J, Okada Y, Reber HA, Hines OJ.Growth stimulation of COX-2-negative pancreatic cancer by a selectiveCOX-2 inhibitor. Cancer Res 65: 982–990, 2005.

13. Feng WY, Liu FT, Patwari Y, Agrawal SG, Newland AC, Jia L.BH3-domain mimetic compound BH3I-2� induces rapid damage to theinner mitochondrial membrane prior to the cytochrome c release frommitochondria. Br J Haematol 121: 332–340, 2003.

14. Green DR, Kroemer G. The pathophysiology of mitochondrial celldeath. Science 305: 626–629, 2004.

15. Gschwendt M, Muller HJ, Kielbassa K, Zang R, Kittstein W, RinckeG, Marks F. Rottlerin, a novel protein kinase inhibitor. Biochem BiophysRes Commun 199: 93–98, 1994.

16. Guha S, Eibl G, Kisfalvi K, Fan RS, Burdick M, Reber H, Hines OJ,Strieter R, Rozengurt E. Broad-spectrum G protein-coupled receptorantagonist, [D-Arg1,D-Trp5,7,9,Leu11]SP: a dual inhibitor of growth andangiogenesis in pancreatic cancer. Cancer Res 65: 2738–2745, 2005.

17. Guha S, Rey O, Rozengurt E. Neurotensin induces protein kinaseC-dependent protein kinase D activation and DNA synthesis in humanpancreatic carcinoma cell line PANC-1. Cancer Res 62: 1632–1640, 2002.

18. Hao JH, Yu M, Liu FT, Newland AC, Jia L. Bcl-2 inhibitors sensitizetumor necrosis factor-related apoptosis-inducing ligand-induced apoptosisby uncoupling of mitochondrial respiration in human leukemic CEM cells.Cancer Res 64: 3607–3616, 2004.

19. Hockenbery DM. A mitochondrial Achilles’ heel in cancer? Cancer Cell2: 1–2, 2002.

20. Hotz HG, Reber HA, Hotz B, Yu T, Foitzik T, Buhr HJ, Cortina G,Hines OJ. An orthotopic nude mouse model for evaluating pathophysi-ology and therapy of pancreatic cancer. Pancreas 26: e89–e98, 2003.

21. Hudson TS, Hartle DK, Hursting SD, Nunez NP, Wang TT, YoungHA, Arany P, Green JE. Inhibition of prostate cancer growth bymuscadine grape skin extract and resveratrol through distinct mechanisms.Cancer Res 67: 8396–8405, 2007.

23. Kamo N, Muratsugu M, Hongoh R, Kobatake Y. Membrane potential ofmitochondria measured with an electrode sensitive to tetraphenyl phospho-nium and relationship between proton electrochemical potential and phos-phorylation potential in steady state. J Membr Biol 49: 105–121, 1979.

24. Kim H, Rafiuddin-Shah M, Tu HC, Jeffers JR, Zambetti GP, Hsieh JJ,Cheng EH. Hierarchical regulation of mitochondrion-dependent apoptosis byBCL-2 subfamilies. Nat Cell Biol 8: 1348–1358, 2006.

25. Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, Schlesinger PH.Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAXinto pores that result in the release of cytochrome c. Cell Death Differ 7:1166–1173, 2000.

26. Kowaltowski AJ, Vercesi AE, Fiskum G. Bcl-2 prevents mitochondrialpermeability transition and cytochrome c release via maintenance ofreduced pyridine nucleotides. Cell Death Differ 7: 903–910, 2000.

27. Kresty LA, Morse MA, Morgan C, Carlton PS, Lu J, Gupta A,Blackwood M, Stoner GD. Chemoprevention of esophageal tumorigen-esis by dietary administration of lyophilized black raspberries. Cancer Res61: 6112–6119, 2001.

28. Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA,Green DR, Newmeyer DD. BH3 domains of BH3-only proteins differ-entially regulate Bax-mediated mitochondrial membrane permeabilizationboth directly and indirectly. Mol Cell 17: 525–535, 2005.

28a.Lee JK, Edderkaoui M, Truong P, Ohno I, Jang KT, Berti A, PandolSJ, Gukovskaya AS. NADPH oxidase promotes pancreatic cancer cellsurvival via inhibiting JAK2 dephosphorylation by tyrosine phosphatases.Gastroenterology 133: 1637–1648, 2007.

29. Mizuta T, Shimizu S, Matsuoka Y, Nakagawa T, Tsujimoto Y. ABax/Bak-independent mechanism of cytochrome c release. J Biol Chem282: 16623–16630, 2007.

31. Ni H, Ergin M, Tibudan SS, Denning MF, Izban KF, Alkan S. Proteinkinase C-delta is commonly expressed in multiple myeloma cells and itsdownregulation by rottlerin causes apoptosis. Br J Haematol 121: 849–856,2003.

32. Reyland ME, Anderson SM, Matassa AA, Barzen KA, Quissell DO.Protein kinase C delta is essential for etoposide-induced apoptosis insalivary gland acinar cells. J Biol Chem 274: 19115–19123, 1999.

33. Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation duringapoptosis. Nat Rev Mol Cell Biol 5: 897–907, 2004.

34. Ringshausen I, Schneller F, Bogner C, Hipp S, Duyster J, Peschel C,Decker T. Constitutively activated phosphatidylinositol-3 kinase (PI-3K)is involved in the defect of apoptosis in B-CLL: association with proteinkinase Cdelta. Blood 100: 3741–3748, 2002.

35. Satoh A, Gukovskaya AS, Nieto JM, Cheng JH, Gukovsky I, Reeve JRJr, Shimosegawa T, Pandol SJ. PKC-� and -ε regulate NF-kappaBactivation induced by cholecystokinin and TNF-� in pancreatic acinarcells. Am J Physiol Gastrointest Liver Physiol 287: G582–G591, 2004.

36. Soltoff SP. Rottlerin is a mitochondrial uncoupler that decreases cellularATP levels and indirectly blocks protein kinase Cdelta tyrosine phosphor-ylation. J Biol Chem 276: 37986–37992, 2001.

37. Stefanelli C, Bonavita F, Stanic I, Farruggia G, Falcieri E, Robuffo I,Pignatti C, Muscari C, Rossoni C, Guarnieri C, Caldarera CM. ATPdepletion inhibits glucocorticoid-induced thymocyte apoptosis. Biochem J322: 909–917, 1997.

38. Sultana A, Tudur Smith C, Cunningham D, Starling N, Tait D,Neoptolemos JP, Ghaneh P. Systematic review, including meta-analyses,on the management of locally advanced pancreatic cancer using radiation/combined modality therapy. Br J Cancer 96: 1183–1190, 2007.

39. Tapia JA, Jensen RT, Garcia-Marin LJ. Rottlerin inhibits stimulatedenzymatic secretion and several intracellular signaling transduction path-ways in pancreatic acinar cells by a non-PKC-delta-dependent mechanism.Biochim Biophys Acta 1763: 25–38, 2006.

40. Tillman DM, Izeradjene K, Szucs KS, Douglas L, Houghton JA. Rottlerinsensitizes colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via uncoupling of the mitochondria inde-pendent of protein kinase C. Cancer Res 63: 5118–5125, 2003.

G72 ROTTLERIN, Bcl-2 PROTEINS, AND APOPTOSIS

AJP-Gastrointest Liver Physiol • VOL 298 • JANUARY 2010 • www.ajpgi.org

on February 12, 2010

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41. Vaquero EC, Edderkaoui M, Nam KJ, Gukovsky I, Pandol SJ,Gukovskaya AS. Extracellular matrix proteins protect pancreatic cancercells from death via mitochondrial and nonmitochondrial pathways. Gas-troenterology 125: 1188–1202, 2003.

42. Vaquero EC, Edderkaoui M, Pandol SJ, Gukovsky I, Gukovskaya AS.Reactive oxygen species produced by NAD(P)H oxidase inhibit apoptosisin pancreatic cancer cells. J Biol Chem 279: 34643–34654, 2004.

43. Warburg O. The Metabolism of Tumors. London, UK: London Constable,1930.

44. Warburg O. On the origin of cancer cells. Science 123: 309–314, 1956.45. Warburg OP, Neigelein K, E. Metabolism of carcinoma cells. Biochem

Z 152: 309–344, 1924.

46. Willis SN, Adams JM. Life in the balance: how BH3-only proteins induceapoptosis. Curr Opin Cell Biol 17: 617–625, 2005.

46a.Yazbec M, Nam KJ, Vaquero EC, Gukovskaya AS, Pandol SJ. Theprotein kinase C delta inhibitor rottlerin greatly stimulates apoptosis inpancreatic cancer cells. Gastroenterology 124: A103, 2003.

47. Zamaraeva MV, Sabirov RZ, Maeno E, Ando-Akatsuka Y, BessonovaSV, Okada Y. Cells die with increased cytosolic ATP during apoptosis: abioluminescence study with intracellular luciferase. Cell Death Differ 12:1390–1397, 2005.

48. Zhang H, Nimmer P, Rosenberg SH, Ng SC, Joseph M. Developmentof a high-throughput fluorescence polarization assay for Bcl-x(L). AnalBiochem 307: 70–75, 2002.

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