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Lupeol, a fruit and vegetable based triterpene, induces apoptotic death of humanpancreatic adenocarcinoma cells via inhibition of Ras signaling pathway

Mohammad Saleemy, Satwinderjeet Kaury,Mee-Hyang Kweony, Vaqar Mustafa Adhami,Farrukh Afaq and Hasan Mukhtar�

Department of Dermatology, University of Wisconsin, Madison, WI, USA

�To whom correspondence should be addressed at Department ofDermatology, University of Wisconsin, 1300 University Avenue, MedicalSciences Center, B-25, Madison, WI, 53706, USA. Tel: þ1 608 263 3927,Fax: þ1 608 263 5223.Email: [email protected]

Pancreatic cancer is an exceptionally aggressive disease,the treatment of which has largely been unsuccessful dueto higher resistance offered by pancreatic cancer cells toconventional approaches such as surgery, radiation and/orchemotherapy. The aberration of Ras oncoprotein has beenlinked to the induction of multiple signaling pathways andto the resistance offered by pancreatic cancer cells to apop-tosis. Therefore, there is a need for development of new andeffective chemotherapeutic agents which can target mul-tiple pathways to induce responsiveness of pancreatic can-cer cells to death signals. In this study, human pancreaticadenocarcinoma cells AsPC-1 were used to investigate theeffect of Lupeol on cell growth and its effects on the modu-lation of multiple Ras-induced signaling pathways. Lupeolcaused a dose-dependent inhibition of cell growth asassessed by MTT assay and induction of apoptosis asassessed by flow cytometry, fluorescence microscopy andwestern blotting. Lupeol treatment to cells was found tosignificantly reduce the expression of Ras oncoprotein andmodulate the protein expression of various signalingmolecules involved in PKCa/ODC, PI3K/Akt andMAPKs pathways along with a significant reduction in theactivation of NFkB signaling pathway. Our data suggestthat Lupeol can adopt a multi-prong strategy to targetmultiple signaling pathways leading to induction of apop-tosis and inhibition of growth of pancreatic cancer cells.Lupeol could be a potential agent against pancreatic can-cer, however, further in-depth in vivo studies arewarranted.

Introduction

Pancreatic cancer is the most fatal of all cancers and the fifthmost common cause of cancer-related deaths among both menand women in the western countries. It is estimated that 32 180cases of pancreatic cancer will be diagnosed in the United

States in 2005 and almost the same number of pancreaticcancer-related deaths are projected (1). Since the mortalityfrom pancreatic cancer compares strikingly with its incidence,it has become a significant public health concern (1). Long-term survival of patients with organ-confined disease is only15%, and in the majority of cases with invasive metastasissurvival is only 4%. Despite these disappointing statistics andthe increase in incidence of pancreatic cancer over the pastseveral decades, the molecular and biochemical determinantsof the disease remain poorly understood and no effectivetherapeutic regimens to significantly ameliorate the clinicalcourse or prognosis of this disease is apparent (1).Thus, pancreatic cancer presents a clinical and experimental

challenge because of its relative resistance to conventionalmodes of therapies such as chemotherapy and radiation andonly 4% of such patients are reported to survive after surgery(1). It is therefore necessary to intensify our efforts for a betterunderstanding of this disease and for the development of novelapproaches for its prevention and treatment. In more than90% of pancreatic cancers, the ras oncogene has been shownto be mutated [(2,3) and references therein]. This mutation isconsidered an early genetic event in the development ofpancreatic cancer and results in constitutive activation of anintracellular pathway leading to cellular proliferation. Severalras-induced signaling pathways, such as the phosphatidylino-sitol 3-kinase (PI3K)/Akt (protein kinase B pathway) mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B(NFkB) pathways, have been linked to chemo- resistance ofthe pancreatic carcinoma cells (3–6). These findings suggestthat Ras oncoprotein could be an important target for devel-oping agents against pancreatic cancer.Epidemiological studies have found a lower risk of pancre-

atic cancer in populations with higher consumption of freshfruits, vitamin C and dietary fiber (7,8). In addition, consump-tion of white wine has been reported to have an inverse relationwith the risk of pancreatic cancer (9). These data suggest thatsome dietary substances could be effective in reducing pan-creatic cancer incidence. We argued that those agents, whichhave the ability to intervene at more than one critical pathwayin pancreatic carcinogenic process, will have greater advant-age over other single-target agents. Lupeol, a triterpene(Figure 1A) is found in various edible plants such as olive,fig, mango, strawberry, red grapes and medicinal plants usedby native people in North America, Japan, China, LatinAmerica and Caribbean islands (10–12). Lupeol has beenshown to exhibit strong anti-inflammatory, anti-arthritic,anti-mutagenic and anti-malarial activity in in vitro andin vivo systems (10). Lupeol has been shown to act as a potentinhibitor of protein kinases and serine proteases and of DNAtopoisomerase II, a target for anticancer chemotherapy(13,14). Lupeol has been reported to induce differentiationand inhibit the growth of melanoma and leukemia cells(15–17). Recently, we have shown that Lupeol inhibitstumor promotion in two-stage mouse skin carcinogenesis by

Abbreviations: DMSO, dimethyl sulfoxide; MAPKs, mitogen-activatedprotein kinases; NFkB, nuclear factor kappa B; ODC, ornithinedecarboxylase; PARP, poly(ADP-ribose)polymerase; PI, propidium iodide;PI3K, phosphatidylinositol 3-kinase; PKCa, protein kinase Ca; PMSF,phenylmethylsulfonyl fluoride; TdT, terminal deoxynucleotidyl transferase.

yThese three authors contributed equally to this work.

Carcinogenesis vol.26 no.11 # Oxford University Press 2005; all rights reserved. 1956

Carcinogenesis vol.26 no.11 pp.1956–1964, 2005doi:10.1093/carcin/bgi157Advance Access publication June 15, 2005

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modulating various signaling pathways (18). In the presentstudy, employing human pancreatic adenocarcinoma cell lineAsPC-1, we show that Lupeol is a potent multi-target antican-cer agent that induces apoptotic cell death of pancreatic cancercells via modulation of Ras-induced protein kinase Ca(PKCa)/ornithine decarboxylase (ODC), PI3K/Akt, MAPKsand NFkB signaling pathways. We suggest that Lupeol may bean effective agent that should be tested for its in vivo efficacyagainst pancreatic cancer.

Materials and methods

Cell culture and reagents

Human pancreatic adenocarcinoma cells AsPC-1 were a gift from Dr NihalAhmad, Department of Dermatology, University of Wisconsin-Madison. Thecells were cultured in RPMI 1640 (ATCC, Manassas VA) in a humidifiedatmosphere of 95% air and 5% CO2 at 37

�C. Antibodies were procured fromBD Transduction laboratories (San Jose, CA), Santacruz Biotechnology (SantaCruz, CA) and Upstate (Charlottesville, VA). Lupeol was purchased fromSigma Chemical Company (St Louis, MO).

Treatment of cells

Lupeol stock (30 mM) was prepared by dissolving Lupeol in warm alcohol anddiluting in dimethyl sulfoxide (DMSO) in a 1:1 ratio. Working solutions wereprepared by diluting the stock solution with DMSO to get desired final con-centrations. For dose-dependent studies, the cells (50% confluent) were treatedwith Lupeol (30, 40 and 50 mM) for 48 h in complete cell medium. Cells thatserved as controls were incubated with the vehicle (alcohol þ DMSO) alone.The final concentration of DMSO and alcohol was 0.25 and 0.075%,respectively, in all treatment protocols.

Cell viability assay

The effect of Lupeol on the viability of AsPC-1 cells was determined byMTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) assay.The cells were plated at 1 � 104 cells per well in 200 ml of complete culturemedium and treated with 10, 20, 30, 40, 50, 75, 100, 125 mM concentrations ofLupeol in 96-well microtiter plates. Lupeol stock solutions prepared at 30 mMconcentration were mixed with fresh medium to achieve the desired finalconcentration. Each concentration of Lupeol was repeated in 10 wells. Afterincubation for 24, 48 and 72 h at 37�C in a humidified incubator, cell viability

was determined. MTT [5 mg/ml in phosphate-buffered saline (PBS)] wasadded to each well and incubated for 2 h after which the plate was centrifugedat 1800 r.p.m. (500� g) for 5 min at 4�C. The supernatant was removed fromthe wells by aspiration. After careful removal of the medium, 0.1 ml ofbuffered DMSO was added to each well, and plates were shaken. The absorb-ance was recorded on a microplate reader at the wavelength of 540 nm. Theeffect of Lupeol on growth inhibition was assessed as percent cell viabilitywhere vehicle-treated cells were taken as 100% viable.

Detection of apoptosis and necrosis by fluorescence microscopy

The Annexin-V-FLUOS apoptosis detection kit (Roche Applied Science,Indianapolis, IN) was used for the detection of apoptotic and necrotic cells.This kit uses a dual-staining protocol in which the cells are stained withannexin and propidium iodide (PI). Cell populations that potentially may bedetected are as follows: viable cells will be non-fluorescent; cells in themetabolically active stages of apoptosis will stain with annexin (green fluor-escence) but not with PI (red fluorescence) and necrotic cells with PI staining.In addition, cells undergoing late stage apoptosis bind both annexin and PI.Briefly, AsPC-1 cells were grown to �50% confluence on slides and thentreated with Lupeol (30, 40, 50 mM) for 48 h. Apoptosis and necrosis wasdetected by the using a Zeiss Axiovert 100 microscope. Briefly, the sampleswere excited at 330–380 nm, and the image was observed and photographedunder a combination of a 400 nm dichoric mirror and then 420 nm high-passfilter.

Quantification of apoptosis

Apoptosis was quantified by terminal deoxynucleotidyl transferase-mediateddUTP-biotin nick and labeling (TUNEL) method. Briefly, the cells were grownat a density of 1 � 106 cells in 100-mm culture dishes and then treated withLupeol (30, 40 and 50 mM) for 48 h. The cells were trypsinized, washed withPBS and processed for labeling with fluorescein-tagged deoxyuridine triphos-phate nucleotide and PI by use of an APO-DIRECT apoptosis kit obtainedfrom Phoenix Flow Systems (San Diego, CA) and following the manufac-turer’s protocol. The labeled cells were then analyzed by flow cytometry.

Total cell lysate and nuclear lysate preparation

The total cell lysate was prepared in cold buffer [50 mM Tris–HCl, 150 mMNaCl, 1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP-40, 1% TritonX-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4] with freshlyadded protease inhibitor cocktail (Protease inhibitor Cocktail Set III; Calbio-chem, La Jolla, CA) in ice. For the preparation of nuclear fractions, the cellswere washed with cold PBS and suspended in 0.4 ml of lysis buffer (10 mMHEPES, pH 7.9, 10 mM KCI, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT,

Lupeol

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Fig. 1. (A) Structure of Lupeol [Lup-20(29)-en-3b-ol], a triterpene is found in fruits and vegetables. (B) Effect of Lupeol on viability of AsPC-1 cells: The cellswere treated with specified concentrations of Lupeol for 24, 48 and 72 h, and cell viability was determined byMTT assay. The values are represented as the percentviable cells where vehicle-treated cells were regarded as 100% viable. Data represent mean value of percent viable cells � SE of three independent experiments.The details are described under Materials and methods.

Lupeol targets multiple signaling pathways

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0.5 mM PMSF, Protease Inhibitor Cocktail Set III) in a microfuge tube. Thecells were incubated on ice for 15 min, after which 12.5 ml of 10% NonidetP-40 was added and the contents were mixed on a vortex and then centrifugedfor 1 min at 4�C at 14 000 g. The nuclear pellet was resuspended in 25 ml ofice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mMEDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, Protease Inhibitor CocktailSet III) and incubated on ice for 30 min with intermittent mixing. The tube wascentrifuged for 5 min at 14 000 g at 4�C and the supernatant (nuclear extract)was stored at�80�C. The protein concentration was determined by BCA assay(Pierce, Rockford, IL) as per the manufacturer’s protocol.

Western blot analysis

For western blot analysis, 40 mg of the protein was resolved over 8–12%polyacrylamide gels and transferred to a nitrocellulose membrane. The blotcontaining the transferred protein was kept in blocking buffer (5% nonfatdry milk, 1% Tween-20; in 20 mM TBS, pH 7.6) on a shaker for 1 h at roomtemperature followed by incubation with appropriate primary antibody inblocking buffer for 1 h to overnight at 4�C. This was followed by incubationwith anti-mouse or anti-rabbit secondary antibody conjugated with horse-radish peroxidase (Amersham Life Sciences) for 1 h. After several washings,immunoblots were detected by chemiluminescence ECL kit (Amersham LifeSciences) and autoradiography using XAR-5 film obtained from EastmanKodak (Rochester, NY). Immunoblots were scanned by HP PrecisionscanPro 3.13 (Hewlett-Packard, Palo Alto, CA). Densitometry measurementsof the scanned bands were performed using digitalized scientific softwareprogram UN-SCAN-IT (Silk Scientific Corporation, Orem, UT). Data werenormalized to b-actin and expressed as mean values� SE of three separate setsof experiments.

Electrophoretic mobility shift assay

To detect NFkB-DNA binding, electrophoretic mobility shift assay (EMSA)was performed using lightshift� chemiluminiscent kit (Pierce, Rockford, IL)by following the manufacturer’s protocol. To start with, DNA was biotinlabeled using the biotin 30 end labeling kit (Pierce, Rockford, IL). Briefly, ina 50 ml reaction buffer, 5 pmol of double-stranded NFkB oligonucleotide 50-AGT TGA GGG GAC TTT CCC AGG C-30; 30-TCA ACT CCC CTG AAAGGG TCC G-50, 10 ml of 5� TdT (terminal deoxynucleotidyl transferase)buffer, 5 ml of 5 mM biotin-N4-CTP, 10 U of diluted TdT, and 25 ml ofultrapure water were incubated at 37�C for 30 min. For supershift assay, thereaction mixture was incubated for an additional 20 min with or withoutantibody for NFkB/p65 (1 ml, Geneka Biotechnology). The reaction wasstopped with 2.5 ml of 0.2 M EDTA. To extract labeled DNA, 50 ml ofchloroform:isoamyl alcohol (24:1) was added to each tube and centrifugedbriefly at 13 000 g. The top aqueous phase containing the labeled DNA wasremoved and saved for binding reactions. Each binding reaction contained 1�-binding buffer (100 mM Tris, 500 mM KCl, 10 mM DTT, pH 7.5), 2.5%glycerol, 5 mM MgCl2, 50 ng/ml poly(dI–dC), 0.05% NP-40, 5 mg of nuclearextract and 20–50 fmol of biotin-end labeled target DNA. The content wasincubated at room temperature for 20 min. To this reaction mixture, 5 ml of5� loading buffer was added, subjected to gel electrophoresis on a nativepolyacrylamide gel and transferred to a nylon membrane. When the transferwas complete, DNA was cross-linked to the membrane at 120 mJ/cm2 using aUV cross-linker equipped with 254 nm bulbs. The biotin end-labeled DNAwasdetected using streptavidin–horseradish peroxidase conjugate and a chemilu-minescent substrate. The membrane was exposed to X-ray film (XAR-5Amersham Life Science, Arlington Height, IL) and developed using a Kodakfilm processor.

Results

Lupeol induces growth inhibition and apoptosis in AsPC-1cells

We first evaluated the effect of Lupeol on cell viability byMTT assay. As shown in Figure 1B, treatment of AsPC-1 cellswith Lupeol (10–125 mM) resulted in a dose-dependent inhibi-tion of cell growth when assessed at 24, 48 or 72 h post-treatment (Figure 1B). The IC50 value of Lupeol on growthinhibition was estimated to be 35 mM at 48 h. Based onthese observations, we selected a dose of 30, 40 and 50 mMand a time period of 48 h post-Lupeol treatment for furthermechanistic studies.We next determined whether Lupeol-mediated loss of cell

viability in AsPC-1 cells is a result of induction of apoptosis.The induction of apoptosis by Lupeol (30–50 mM) was evident

from the morphology of cells, as assessed by fluorescencemicroscopy after labeling the cells with annexin and PI(Figure 2, upper panel). As shown by representative pictures(Figure 2, upper panel), Lupeol treatment resulted in inductionof apoptosis in a dose-dependent manner. These data also showthat Lupeol treatment also resulted in necrosis of these cells,which may be a secondary event in the apoptotic process. Wenext quantified the extent of apoptosis by flow-cytometricanalysis of Lupeol-treated cells labeled with bromo-deoxyuridine triphosphate and PI. As shown by the data inFigure 2 (bottom panel), compared with control, Lupeol treat-ment resulted in 0.3, 8.6 and 22.0% TUNEL-positive cellsat 30, 40 and 50 mM at 48 h post-treatment, respectively.Consistent with the fluorescence microscopy data, TUNELassay by flow cytometry revealed that treatment of AsPC-1cells with Lupeol resulted in a dose-dependent induction ofapoptosis.

Lupeol induces PARP cleavage and increases Bax proteinexpression in AsPC-1 cells

Since poly(ADP-ribosylation) is a post-translational modifica-tion of proteins which plays a crucial role in DNA repair andcell death, poly(ADP-ribose)polymerase (PARP), a DNA nicksensor, serves as one of the best-known biomarkers of apop-tosis (19). During apoptosis, PARP protein is cleaved intoan 85 kDa C-terminal fragment, with a reduced catalytic activ-ity, and a 24 kDa N-terminal peptide, which retains the DNAbinding domains. Next, we assessed the effect of Lupeol(30–50 mM) treatment of cells on PARP cleavage. ThePARP cleavage analysis showed that the full-size PARP(116 kDa) protein was cleaved to yield an 85 kDa fragmentafter treatment of cells with Lupeol at 30, 40 and 50 mMconcentrations at 48 h post-treatment (Figure 3). The expres-sion of Bax (pro-apoptotic protein) and Bcl-2 (anti-apoptoticprotein) have been reported to play a crucial role in apoptoticresponse mediated by many agents (20,21). We next determ-ined the effect of Lupeol treatment of cells on the proteinlevels of Bax and Bcl-2. The immunoblot analysis exhibiteda significant increase in the protein expression of Bax inLupeol-treated cells (Figure 3). However, the protein expres-sion of Bcl-2 did not exhibit any change by Lupeol treatment atany dose.

Lupeol induces activation of caspases in AsPC-1 cells

Caspases receive the upstream death signals and are the finalexecutioners of apoptosis (22,23). In most cancer cells,caspases are present in the pro-forms (inactive) and requirecleavage of protein to become active to participate in theprocess of apoptosis. Through this process, the pro-forms ofcaspases are lost and the expression of their active forms(cleaved products) increases during apoptosis (22,23). Wenext performed immunoblot analysis of the members of cas-pases in the cell lysates obtained from Lupeol-treated cells. Wehave used antibodies that specifically recognize the pro-formsand cleaved products (active forms) of caspases. As shown inFigure 4, Lupeol-treated cells did not exhibit any change in theexpression level of procaspase-3, however, the expressionof active caspase-3 was found to be increased in such cells(Figure 4). Further Lupeol treatment induced the expression ofcaspases-8 and -9 in a dose-dependent manner which is evidentfrom the decrease in the protein expression of their pro-forms(inactive forms) with a concomitant increase in their activeforms as compared with vehicle-treated control (Figure 4).

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Lupeol decreases the expression of Ras, PKCa, ODC proteinsand inhibits PI3K/Akt in AsPC-1 cells

Many cancers including pancreatic cancer exhibit aberrant Rassignaling that leads to chronic up-regulation of the Ras path-way and accumulation of Ras oncoprotein in transformedcells (24). PKCa has been implicated in Ras signaling andtransformed cells have been reported to have high levels ofPKCa (25). In various cancer types, Ras-induced PKCa hasbeen reported to activate ODC, an oncogene which is highlyactivated during the cellular proliferation (25–27). Next, wedetermined the effect of Lupeol treatment of cells on theexpression of Ras, PKCa and ODC proteins. It is evidentfrom the data of immunoblot analysis that treatment of Lupeolsignificantly reduced the protein expression of Ras in a dose-dependent manner (Figure 5). Similarly, treatment of Lupeolto cells also caused a significant reduction in the proteinexpression of ODC and PKCa (Figure 5). These findingssuggest that Lupeol exerts a preferential inhibitory effect onRas/PKCa/ODC pathway leading to reduction in pancreaticcancer cell growth.The frequent aberration in Ras signaling is reported to cause

constitutive activation of PI3K/Akt during the development ofpancreatic cancer in humans [(6,28) and references therein].We next investigated the effect of Lupeol treatment of cells onPI3K/Akt signaling pathway. Lupeol treatment of cells wasfound to cause a significant reduction in the expression ofregulatory subunit of PI3K (p85) protein (Figure 5). Similarly,Lupeol caused a significant dose-dependent decrease in thephosphorylation of Akt protein in cells (Figure 5). The treat-ment of Lupeol did not cause any change in the protein levelsof total Akt (Figure 5). These results suggest the inhibitorypotential of Lupeol against PI3K/Akt pathway, which is highly

Fig. 2. Lupeol induces apoptosis in AsPC-1 cells as assessed by fluorescence microscopy (upper panel) and by flow cytometry (bottom panel). The upperpanel contains the representative micrographs of AsPC-1 cells undergoing apoptosis induced by treatment with Lupeol as assessed by fluorescence microscopy.The cells were treated with vehicle alone or specified concentration of Lupeol for 48 h. Apoptosis and necrosis was detected by a Zeiss Axiovert 100 microscopeas described in Materials and methods. The samples were excited at 330–380 nm, and the image was observed and photographed under a combination of a400 nm dichoric mirror and then 420 nm high pass filter. The bottom panel is the quantitative representation of Lupeol-induced apoptosis in AsPC-1 cellsas assessed by flow cytometry. The cells were treated with Lupeol (30–50 mM for 48 h), labeled with deoxyuridine triphosphate using terminal deoxynucleotidetransferase and PI by using an apoptosis APO-DIRECT kit obtained from Phoenix Flow Systems (San Diego, CA) as per vendor’s protocol followed byflow cytometry. Cells showing deoxyuridine triphosphate fluorescence above that of control population, as indicated by the line in each histogram, are consideredas apoptotic cells. The images shown here are representative of three independent experiments with similar results. The values shown inside each graph indicatethe percent apoptotic cells. The details are described under Materials and methods.

Fig. 3. Effect of Lupeol treatment on PARP cleavage and on proteinexpression of Bax, Bcl-2 in AsPC-1 cells. The cells were treated with vehicle(DMSO þ alcohol) only or specified concentrations of Lupeol for 48 h,harvested and total cell lysates were prepared. PARP cleavage and theexpression of Bax and Bcl-2 were determined by western blot analysis. Equalloading was confirmed by stripping immunoblots and reprobing them forb-actin. Values given above each lane on the immunoblot represents therelative density of the bands normalized to b-actin. The immunoblots shownhere are representative of three independent experiments with similar results.The details are described under Materials and methods.


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activated during the development and progression of pancre-atic cancer.

Lupeol inhibits the expression of MAPK proteins p38 andErk1/2 in AsPC-1 cells

Constitutive levels of Erk1/2 protein have been reported to bevery low in pancreatic cancer cells and studies have linked thedysregulation of MAPKs with tumor cell survival in variouscancer types including pancreatic adenocarcinoma (3,29,30).Therefore, we determined whether MAPKs contributed toLupeol-induced apoptosis in our model. We addressed thisquestion by determining the effect of Lupeol treatment onexpression levels of Erk1/2, JNK and p38 MAPK proteins.As shown in Figure 5, exposure of cells to Lupeol resulted in adose-dependent activation (phosphorylation) of Erk1/2.Lupeol treatment caused a dose-dependent decrease inthe level of phospho-p38 MAPK (Figure 5). Unlike ERK1/2and p38, however, Lupeol treatment did not alter the level of

JNK1 protein (data not shown). Taken together, these obser-vations indicate that activation of Erk1/2 protein and decreasein expression levels of phospho-p38 protein might be contrib-uting towards apoptosis of cells induced by Lupeol.

Lupeol inhibits phosphorylation of IkBa and NF-kB/p65 inAsPC-1 cells

A number of studies clarified the role of NFkB in tumor cellsurvival by inhibiting apoptosis (31). The activation of NFkB

Fig. 4. Effect of Lupeol treatment on protein expression of caspases-3, -8 and-9 in AsPC-1 cells. The cells were treated with vehicle (DMSO þ alcohol)only or specified concentrations of Lupeol for 48 h, harvested and total celllysates were prepared. The expression of proforms and cleaved products ofcaspase -3, -8 and -9 were determined by western blot analysis. Equalloading was confirmed by stripping immunoblots and reprobing them forb-actin. Values given above each lane on the immunoblot represents therelative density of the bands normalized to b-actin. The immunoblots shownhere are representative of three independent experiments with similar results.The details are described under Materials and methods.

Fig. 5. Effect of Lupeol treatment on the expression of Ras, PKCa, ODC,PI3K (P85), pAkt and total Akt, phospho-p38 and Erk1/2 proteins in AsPC-1cells: the cells were treated with vehicle (DMSOþ alcohol) only or specifiedconcentrations of Lupeol for 48 h, harvested and total cell lysates wereprepared. The expression of Ras, PKCa, ODC, PI3K(P85), pAkt and totalAkt, p38 and Erk1/2 proteins total cell lysate was determined by western blotanalysis. Equal loading was confirmed by stripping immunoblots andreprobing them for b-actin. Values given above each lane on the immunoblotrepresents the relative density of the bands normalized to b-actin. Theimmunoblots shown here are representative of three independentexperiments with similar results. The details are described under Materialsand methods.

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pathway in pancreatic cancer cells has been linked totheir resistance to conventional chemotherapy (32). Thephosphorylation of inhibitory protein IkBa is a criticalstep in the pathway of NFkB activation that results in thetranslocation of NFkB molecule to nucleus from cytosol,which ultimately leads to the survival of tumor cells. Thetranslocation of NFkB to nucleus is preceded by the phos-phorylation of its p65 subunit and therefore is an importantevent. Next, we investigated the effect of Lupeol treatment onthe phosphorylation of IkBa and NFkB/p65 in cells. As shownby immunoblot analysis (Figure 6A), Lupeol treatmentresulted in a dose-dependent inhibition of phosphoryla-tion of NFkB/p65 protein in these cells. The inhibition ofphosphorylation of NF-kB/p65 molecule was concomitantwith a decrease in the phosphorylation of IkBa protein(Figure 6A).

NFkB–DNA binding is reduced in Lupeol-treated AsPC-1cells

The translocation of NFkB to nucleus is marked by its bindingwith DNA to activate the expression of various genes leadingto the tumor cell survival (31). We next performed EMSA toinvestigate the effect of Lupeol treatment on NFkB–DNAbinding activity. As shown in Figure 5B, untreated and vehiclecontrol cells exhibited a constitutively increased NFkB–DNAbinding activity; however, in cells treated with Lupeol, asignificant decrease in NFkB–DNA binding activity wasobserved (Figure 6B). The effect of Lupeol on NFkB–DNAbinding in cells was concomitant with the effect of Lupeol onphosphorylation of NF-kB/p65 subunit (Figure 6A). As isevident from results of supershift assay using anti-p65 anti-bodies, Lupeol was observed to specifically decrease the levelsof p65 subunit of NF-kB in the nucleus of cells (Figure 6C).

Fig. 6. (A) Effect of Lupeol treatment on the phosphorylation of IkBa protein and p65-subunit of NFkB transcriptional factor in AsPC-1 cells. The cells weretreated with vehicle (DMSO þ alcohol) only or specified concentrations of Lupeol for 48 h, harvested and total cell lysates were prepared. Equal loading wasconfirmed by stripping immunoblots and reprobing it for b-actin. Values given above each blot represents the relative density of the bands normalized to b-actin.The immunoblots shown here are representative of three independent experiments with similar results. The details are described under Materials and methods.(B) Effect of Lupeol treatment on the binding of NFkB transcriptional factor with DNA in AsPC-1 cells as assessed by EMSA. After 48 h of treatment withLupeol, the cells were harvested, nuclear lysates were prepared and DNA binding was determined by EMSA as described under Materials and methods.I, II, and III refer to internal experimental controls, where I represents biotin–EBNA (Epstein–Barr virus nuclear antigen) control DNA, II represents biotin–EBNAcontrol DNA and EBNA extract, and III represents biotin–EBNA control DNA and EBNA extract plus 200-fold molar excess of EBNA DNA. In controlnumber I, no protein extract for DNA to bind resulted in an unshifted band. In control number II, sufficient target protein leads to DNA–protein binding resulting inshift detected by comparison to band at position I. Control number II demonstrated that the signal shift observed could be prevented by competition fromexcess unlabelled DNA. The data shown here are representative of three independent experiments with similar results. (C) Effect of Lupeol treatment on thebinding of p65-subunit of NFkB transcriptional factor with DNA in AsPC-1 cells. The cells were treated with vehicle only or specified concentrations ofLupeol for 48 h, and then processed for nuclear lysate. NFkB/DNA binding activity was determined by Supershift assay using anti-p65 antibody and EMSA kitfrom Pierce (Rockford, IL). The details are described under Materials and methods. The data shown here are representative of three independent experimentswith similar results, ns represents non-specific binding.


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Discussion

The aggressive nature of pancreatic adenocarcinoma is relatedto several abnormalities in growth factors and their receptorsthat affect the downstream signal transduction pathwaysinvolved in the control of growth and differentiation (4).These perturbations confer a tremendous survival and growthadvantage to pancreatic cancer cells. The acquisition of anapoptosis-resistant phenotype by pancreatic cancer cells mayalso upset the success of conventional radio- and chemother-apies (32). In addition to perturbations in cell cycle control,resistance to apoptosis may promote tumor growth and meta-stasis. Apoptosis resistance mechanisms seem to vary amongdifferent tumor types and have only begun to be characterizedat the molecular level.Elucidation of molecular events during pancreatic cancer

development has led to several distinct therapeutic advancesand many novel agents and inhibitors including monoclonalantibodies have been developed and are undergoing clinicaltrials (4,32–34). However, many of these agents are not effect-ive alone and have to be combined for maximum efficacyleading to undesirable and sometimes fatal side effects. Onepromising approach could be identification of non-nutrientdietary constituents that can target multiple pathways withoutcausing any undesirable toxicity. Here, we describe the use-fulness of Lupeol, a novel non-nutrient dietary agent againsthuman pancreatic cancer cells. In the current study, we dem-onstrate that Lupeol treatment induces a dose-dependent deathof human pancreatic cancer cells. From our data, it is evidentthat the induction of cell death is mediated through apoptosis.We also found that Lupeol-induced apoptosis of pancreaticcells is mediated through the activation of caspases-3, -8 and-9. The observation that expression levels of procaspase-3 didnot exhibit any significant change upon Lupeol treatmentcould be explained through a possible involvement of a feed-back mechanism, which restores depleted procaspase-3 levelsat intracellular level. The only other pro-apoptotic effect oflupeol reported so far is on mouse melanoma and humanleukemia cells (15–17).Transformed cells have been shown to possess a dysregu-

lated apoptotic machinery (35). Several pro-apoptotic proteinsare either lost or diminished and many anti-apoptotic proteinsare activated during the development of cancer (35). As isevident from our data, induction of pro-apoptotic Bax proteinby Lupeol indicates that Lupeol may rectify the errors inapoptotic machinery of pancreatic cancer cells. Althoughmany studies have shown that natural agents decrease theexpression of anti-apoptotic protein Bcl-2 with a concomitantincrease in the expression of pro-apoptotic Bax protein andshift the balance towards apoptosis, we did not observeany change in the Bcl-2 protein expression in Lupeol-treatedpancreatic cancer cells. However, with an increase in Baxexpression the shift is indeed towards apoptosis. Since ourdata suggest that Lupeol is a multi-target agent, other mech-anisms of apoptosis could not be ruled out.Other contributing molecular changes in pancreatic adeno-

carcinoma include activation of oncogenes and inactivation oftumor suppressor genes (2–5). The ras oncogene is thought toplay an important role in the growth of pancreatic cancer,because an activated (mutated) ras gene is found in �90% ofhuman pancreatic cancers (2–5). The ras mutation results inthe accumulation of Ras oncoprotein, and constitutive activa-tion of various intracellular signaling pathways, leading to

cellular proliferation (2–6). Targeting the ras gene has beenshown to inhibit the growth of metastatic pancreatic cells (4).In the present study, we observed that treatment of humanpancreatic cancer cells with Lupeol decreases the proteinexpression of Ras in a dose-dependent manner.Ras protein is reported to regulate various downstream

signaling pathways such as the PI3K/Akt pathway. Thegrowth-promoting potential of the PI3K/Akt pathway and itsanti-apoptotic properties are closely linked to the resistance ofcancer cells to a broad spectrum of apoptotic stimuli andseveral studies have demonstrated that treatment with thePI3K inhibitors substantially enhances apoptosis (4–6,36,37).Studies have shown that inhibitors of PI3K/Akt induce dose-dependent apoptosis of pancreatic cancer cells and inhibittumor growth of pancreatic cancer xenografts (37,38). In thecurrent study, we show that treatment of pancreatic cancercells with Lupeol reduced the expression of PI3K and Aktproteins in a dose-dependent manner. This is consistent withour earlier report where we demonstrated that Lupeol inhibitedthe PI3K/Akt signaling pathway in vivo during two-stagemouse skin tumorigenesis (18). These results suggest thatLupeol has an efficiency to modulate the signaling pathwayswhich are known to promote pancreatic cancer cell growthand survival.The effects mediated by aberrantly activated Ras in human

cancers are likely to be very complex and are currently notcompletely understood. Both Ras and PI3K/Akt signalingpathways have been reported to regulate the expression ofMAPKs during the progression of various cancer types includ-ing pancreatic cancer (3,4,29,30). Chronic stimulation of theRas/Raf/MEK/ERK pathway by oncogenic Ras is thought topromote cellular transformation and this pathway has beenreported to be activated in various cancer types, however,Erk1/2 activation does not always correlate with the expres-sion of mutant ras or other oncogenes (3). Constitutive levelsof Erk1/2 proteins have been reported to be very low inpancreatic cancer cells despite Ras activation and Erk1/2inhibition in pancreatic cancer cells have been linked withthe resistance to apoptosis [(3, 30) and references therein].As evident from our data, the correlation of significant celldeath with increased expression of Erk1/2 proteins in Lupeol-treated cells is evidence that low levels of Erk1/2 proteinhave an association with tumor cell survival in pancreaticcancer. Our data are supported by recent studies showing thatchemotherapeutic agent-induced apoptosis in pancreatic can-cer cells is through Erk1/2 activation. It has been demonstratedthat p38 MAPK activation triggers an anti-apoptotic responseto tumor cells, although there have also been some contrastingreports that p38 MAPK activation (phosphorylation) is centralto the pro-apoptotic effects in some cancer cell lines (39,40).Recent reports have demonstrated a link between the Rasoncoprotein and p38 MAPK activation in transformed cells(41). It is evident from our data that Lupeol treatment signi-ficantly decreases the expression of phospho-p38 MAPK incells leading to their apoptotic death.Ras oncoprotein has been shown to contribute to constitutive

activation of NFkB signaling in pancreatic cancer through theactivation of PI3K/Akt and MAPK pathways (32). Wang et al.reported that p65 subunit of NFkB was constitutively activatedin �67% of pancreatic adenocarcinomas and a positive cor-relation exists between the activation of NFkB and Rasoncoprotein during the proliferation of pancreatic cancercells (42). NFkB plays an important role in tumor resistance

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to apoptosis and studies have shown that pancreatic cancercells resistant to apoptosis exhibit a high basal NFkB activity(31,43). Interestingly, we found that treatment of Lupeol inhib-ited expression of NFkB and phosphorylation of IkBa proteinin pancreatic cancer cells. Because Lupeol inhibits IkBa phos-phorylation, our study suggests that the effect of Lupeol onNFkB/p65 is through inhibition of phosphorylation of IkBawhich in turn is regulated by Ras and PI3K/Akt pathways.The Ras oncoprotein is also reported to regulate PKCa

(23,24). PKCa has been reported to be involved in the regula-tion of apoptosis and in animal models, tumorigenicity ofpancreatic cancer cells has been directly related to increasedexpression of PKCa protein (25,26). Numerous studies haveshown that PKC levels are high in pancreatic cancer patientsand increased PKC levels offer resistance to apoptosis ofpancreatic cancer cells (32). In this scenario, Lupeol with anefficacy of targeting PKCa protein seems to be a promisingagent against the growth of pancreatic cancer cells as it exhib-its an inhibitory potential against PKCa protein and inducesapoptosis of pancreatic cancer cells in a dose-dependentmanner.Ras-induced PKCa is known to induce the ODC protein,

the first and the rate-limiting enzyme in the biosynthesis ofpolyamines expression during the development of variouscancer types (26,27,44). Elevated levels of ODC gene productsare consistently detected in transformed cell lines, virtuallyall-animal tumors and in certain tissues predisposed to tumori-genesis (24,25). Because tumor formation can be prevented bythe agents that block induction of ODC, several ODC inhibit-ors have been tested against various types of cancers [(18) andreferences therein]. These data demonstrate that Lupeol isa potent inhibitor of ODC expression and is consistent withour earlier report where we showed that Lupeol inhibits ODCactivity and expression in vivo (18).Based on the outcome of this study, we suggest Lupeol could

provide a multi-prong beneficial strategy for targeting multiplesignaling pathways leading to apoptosis and inhibition of

growth of pancreatic cancer cells. The schematic representa-tion of the action of Lupeol is depicted in Figure 7. This maybe explained by modulation of NFkB mediated by Ras-induced pathways such as PKCa/ODC, PI3K/Akt and MAPKsignaling. Since pancreatic cancer is resistant to conventionalchemotherapeutic regimens, Lupeol could be a potential agentto overcome this resistance. Further in-depth in vivo studies arewarranted to verify this suggestion and are presently underinvestigation in our laboratory.

Supplementary material

Supplementary material can be found at: http://carcin.oxfordjournals.org/

Acknowledgements

Author (S.K.) was recipient of BOYSCAST fellowship from Department ofScience and Technology, Government of India. This work was supported byUnited States Public Health Service grants RO1 CA 78809 and RO1 CA101039.

Conflict of Interest Statement: None declared.

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Fig. 7. A schematic representation of the action of Lupeol with respect to various signaling pathways in AsPC-1 cell. " Represents increased expression;# represents decreased expression; ‘ represents inhibition; and ! represents induction. See online Supplementary material for a color version of this figure.


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Received April 12, 2005; revised May 26, 2005; accepted June 7, 2005

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