Guggulsterones induce apoptosis and differentiation in ... · suggest that in addition to its lipid-lowering activity guggulsterone may modulate anti-inflammatory and anti-oxidant

Guggulsterones induce apoptosis and differentiationin acute myeloid leukemia: identification ofisomer-specific antileukemic activities ofthe pregnadienedione structure

Ismael Samudio,1 Marina Konopleva,1

Stephen Safe,2,3 Teresa McQueen,1

and Michael Andreeff1

1Section of Molecular Hematology and Therapy, Department ofBlood and Marrow Transplantation, The University of TexasM.D. Anderson Cancer Center; 2Institute of Biosciences andTechnology, Texas A&M University, Houston, Texas and3Department of Veterinary Physiology and Pharmacology, TexasA&M University, College Station, Texas

AbstractIn this study, the antileukemic effects of three isomericpregnadienedione steroids [i.e., cis -guggulsterone,trans -guggulsterone, and 16-dehydroprogesterone]were investigated in HL60 and U937 cells as well asin primary leukemic blasts in culture. Our results showthat all three compounds inhibited the proliferation ofHL60 and U937 cells, with IC50s ranging from 3.6 to10.9 Mmol/L after treatment for 6 days. These growthinhibitory effects correlated with externalization ofphosphatidylserine and loss of mitochondrial membranepotential, suggesting that these isomeric steroids induceapoptosis in leukemia cells. z-VAD-fmk prevented phos-phatidylserine externalization but not mitochondrialmembrane potential loss, indicating that mitochondrialdysfunction occurred in the absence of caspase acti-vation. Interestingly, although all three compoundsincreased the generation of reactive oxygen speciesand decreased phosphorylation of extracellular signal-regulated kinase, only cis-guggulsterone induced a rapiddepletion of reduced glutathione levels and oxidation ofthe mitochondrial phospholipid cardiolipin. 16-Dehydro-progesterone and trans-guggulsterone induced differen-tiation of HL60 and NB4 cells as evidenced by increasedsurface expression of CD11b and/or CD14, and all threesteroids rapidly induced mitochondrial dysfunction and

phosphatidylserine externalization of CD34-positiveblasts from primary leukemic samples. This study is thefirst to show that guggulsterones and 16-dehydroproges-terone exert antileukemic effects via the induction ofapoptosis and differentiation and, more importantly,identifies the pregnadienedione structure as a potentialchemotherapeutic scaffold. [Mol Cancer Ther 2005;4(12):1982–92]

IntroductionAcute myeloid leukemias (AML) are clonal malignanciescharacterized by increased numbers of immature myeloidprogenitor cells arrested at different stages of granulocyticand monocytic differentiation. First-line treatment of AMLconsists of a combination of cytarabine and an anthracy-cline, and although this combination results in 60% to 80%complete remissions in newly diagnosed patients, mostpatients will relapse with resistant disease (1). Becauseachievement of complete remission is a prerequisite forlong-term survival (2), several novel therapeutic modalitieshave been investigated, including the use of differentanthracycline formulations, different nucleoside analogues,and the combination of the antiangiogenic agent thalido-mide with cytarabine/anthracycline or topotecan/anthra-cycline (3–5). However, overall improvement in survivalrates has been marginal at best underlining the need fordevelopment of more effective therapies. The most strikingincrease of complete remission and survival has beenachieved by ligation of the nuclear retinoic acid receptor ain acute promyelocytic leukemias with all-trans retinoicacid (6, 7).The gum resin from the guggul tree Commiphora mukul

has been used in Ayurvedic medicine for centuries to treatinflammatory and lipid disorders (8), and an ethylacetateextract of the resin, termed guggulipid, has been reportedto have an antiobesity and antilipidemic effect in clinicaltrials with no significant toxicity (9–12). The activesubstances in guggulipid are the pregnane plant sterolscis-guggulsterone and trans-guggulsterone, which havebeen shown to lower cholesterol and triglycerides innormal and high-fat-fed rats (9). The antilipidemic effectsof guggulsterone may be mediated by antagonism of theorphan receptor FXR (13) as well as promiscuous inter-actions with other nuclear receptors (14). Notably, althoughmost studies on guggulsterone have focused on theirantilipidemic activity, these compounds have also shownpotent anti-inflammatory effects, such as preventingoxidative damage during isoproterenol-induced myocar-dial necrosis in rats (15, 16) and decreasing inflammation

Received 7/18/05; revised 9/14/05; accepted 9/23/05.

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

Requests for reprints: Michael Andreeff, Section of Molecular Hematologyand Therapy, Department of Blood and Marrow Transplantation, TheUniversity of Texas M.D. Anderson Cancer Center, Unit 448, 1400Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-7260;Fax 713-794-474. E-mail: [email protected]

Copyright C 2005 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-05-0247

1982

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on May 1, 2020. © 2005 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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associated with nodulocystic acne (17). These observationssuggest that in addition to its lipid-lowering activityguggulsterone may modulate anti-inflammatory and anti-oxidant responses.A variety of naturally occurring compounds exhibit

chemopreventive and anti-inflammatory effects, includ-ing resveratrol, betulinic acid, saikosaponin, and curcu-min. Some of the chemotherapeutic activities of thesecompounds may be related to their inhibition of nuclearfactor-nB signaling (18–22), and a recent study reportedthat cis-guggulsterone inhibited tumor necrosis factor-a–induced nuclear factor-nB signaling and sensitizedcancer cells to apoptosis induced by taxol, doxorubicin,and tumor necrosis factor-a (23). Surprisingly, there areno studies to date investigating the direct antiprolifer-ative and proapoptotic effects of guggulsterone in cancercell lines in culture. We therefore hypothesized that

guggulsterone, like other anti-inflammatory and chemo-preventive agents, may decrease the proliferation ofcancer cells in culture. Here, we report that both isomersof guggulsterone, cis-guggulsterone and trans-guggul-sterone, effectively inhibit the proliferation of leukemiccancer cell lines and induce apoptosis and differentia-tion. Interestingly, a mammalian steroid metabolite andchemical isomer of guggulsterone, 16-dehydroprogester-one, also induced a comparable pattern of differentiation,growth inhibition, and apoptosis, suggesting that thepregnadienedione structure of these steroids (Fig. 1A)offers the potential for development of novel chemo-therapeutics. Our results are the first to show theantileukemic effects of guggulsterone isomers and 16-dehydroprogesterone, and current studies are investigat-ing their mechanism of action and development of morepotent novel steroidal analogues.

Figure 1. Guggulsterone isomers and 16-dehydroprogesterone prevent the proliferation of HL60 and U937 cells in long-term culture. A, structure of thepregnadienedione isomers used in this study. B, HL60 cells were cultured in the presence of increasing concentrations of the guggulsterone isomers and16-dehydroprogesterone (10–20 Amol/L) for 72 and 144 h. Viable cells were counted using a hemocytometer after trypan blue staining. C, U937 cellswere treated with the guggulsterone isomers and 16-dehydroprogesterone as for HL60 cells above. All experiments were done in duplicate and repeated atleast thrice. cGS, cis -guggulsterone; tGS, trans -guggulsterone; P, 16-dehydroprogesterone. Points, mean of three independent experiments; bars, SE.

Molecular Cancer Therapeutics 1983




Materials andMethodsCell Lines, Chemicals, and BiochemicalsU937 and HL60 cells were maintained in RPMI supple-

mented with 10% FCS, 1% glutamine, and 100 units/mLpenicillin in a 37jC incubator containing 5% CO2. 16-Dehydroprogesterone, cis-guggulsterone, and trans-gug-gulsterone were purchased from Steraloids, Inc. (Newport,RI). TMRM, dihydroethidine, and Cell Tracker Green wereall obtained from Molecular Probes (Eugene, OR). z-VAD-fmk was purchased from Alexis Biochemicals (AxxoraLLC, San Diego, CA). Phospho–extracellular signal-regu-lated kinase (ERK) and total ERK antibodies werepurchased from Cell Signaling Technologies, Inc. (Beverly,MA). Heme oxygenase-1 antibody was purchased from BDBiosciences (San Jose, CA) and a-tubulin was purchasedfrom Santa Cruz Biotechnology (Santa Cruz, CA). All otherchemicals used were of the highest purity available.

Human SubjectsBone marrow or peripheral blood samples were obtained

for in vitro studies from patients with AML. Samples werecollected during routine diagnostic procedures after in-formed consent was obtained in accordance with regu-lations and protocols approved by the Institutional ReviewBoard of The University of Texas M. D. Anderson CancerCenter (Houston, TX). Mononuclear cells were separatedby Ficoll-Hypaque (Sigma Chemical, St. Louis, MO) densitygradient centrifugation. Patient sample 1 was a bonemarrow aspirate containing 85% blasts from an AML-M1relapse patient (�7del). Patient sample 2 was a bonemarrow aspirate containing 95% blasts from an AML-M1relapse patient [t(12,17)]. Patient sample 3 was a bonemarrow aspirate containing 97% blasts from an AML-M2relapse patient (normal cytogenetics). Patient sample 4 wasa peripheral blood sample containing 92% blasts from anAML-M0 relapse patient [�7del; t(11,19)]. To investigatethe effects of the guggulsterone and 16-dehydroprogester-one on normal cells, blood samples from three healthyvolunteers (A-C) were obtained and peripheral bloodmononuclear cells (PBMC) were separated by Ficoll-Hypaque density gradient centrifugation. PBMC sampleswere then exposed to 100 Amol/L guggulsterone and 16-dehydroprogesterone for 20 hours, and phosphatidylserineexternalization was quantitated by flow cytometry.

Measurement of Intracellular Glutathione by FlowCytometryCells (3 � 105/mL; 0.5 mL) were treated with compounds

as indicated or with 2 mmol/L diethylmaleate for 30minutes. Cells were then collected by centrifugation,washed in PBS once, and resuspended in 0.2 mL PBScontaining 400 Amol/L Cell Tracker Green and incubated at20jC protected from light for 10 minutes. Cells were thenwashed in PBS several times, and Cell Tracker Greenfluorescence was quantitated by flow cytometry. The meanCell Tracker Green fluorescence from diethylmaleate-treated samples was considered to be background andsubtracted accordingly. All experiments were done induplicate and repeated at least thrice.

Measurement of Phosphatidylserine ExternalizationandMitochondrial Membrane PotentialAfter appropriate treatments, cells were washed twice in

PBS and then resuspended in 100 AL Annexin bindingbuffer [140 mmol/L NaCl, 10 mmol/L KH2PO4, 5 mmol/LCaCl2 (pH 7.4)] containing 25 nmol/L TMRM and 1:100dilution of Annexin V-FLUOS (Roche Diagnostics,Mannheim, Germany) incubated at 37jC for 30 minutes.Cells were then analyzed by flow cytometry in aFACSCalibur flow cytometer using a 488 nm argon exci-tation laser.

MeasurementofReactiveOxygenSpeciesGenerationAfter appropriate treatments, cells were harvested

by centrifugation, washed in PBS, and loaded with theO2

�-sensitive probe dihydroethidine. Cells were incubated at37jC for 10 minutes and washed in PBS, and FL2 fluo-rescence was examined by flow cytometry. Results pre-sented are means F SE of three independent experiments.

Western Blot AnalysisCells where harvested by centrifugation, washed twice in

PBS, and resuspended in ice cold lysis buffer [1% TritonX-100, 45 mmol/L KCl, 10 mmol/L Tris (pH 7.5)] supple-mented with protease and phosphatase inhibitors and thensubjected to SDS-PAGE in 10% or 12% polyacrylamide gelsfollowed by protein transfer to a Hybond-P membrane(Amersham Pharmacia Biotech, Little Chalfont, UnitedKingdom) and immunoblotting. Signals were detected bya PhosphorImager (Storm 860, version 4.0, MolecularDynamics, Sunnyvale, CA).

Measurement of Cardiolipin ContentAfter appropriate treatments, cells were harvested by

centrifugation, washed once in PBS, and resuspended inPBS containing 10 nmol/L nonyl acridine orange, aprobe that binds with high affinity to reduced but notoxidized cardiolipin (24). Cells were incubated 37jC for30 minutes and FL1 fluorescence was quantitated byflow cytometry.

HL60 and NB4 Cell DifferentiationHL60 and NB4 cells were treated with 10 Amol/L

guggulsterone and 16-dehydroprogesterone, and after120 hours, the cells were collected, washed once inPBS, and resuspended in PBS containing 1:100 dilutionof CD11b-phycoerythrin and CD14-FITC (both form BDBiosciences). Cells were incubated at room temperaturefor 15 minutes, washed in PBS, and analyzed by flowcytometry gating on viable cells as determined byforward and side scatter. Cells stained with mouse IgG-phycoerythrin and mouse IgG-FITC served as negativecontrols. In parallel, viable cells were counted in ahemocytometer after trypan blue exclusion. The absolutenumber of differentiated cells was calculated from theequation:

ð½Viable cells ð1� 104Þ� � ½%CD14 or CD11bþcells�Þ=100%

Results were expressed as the total number of viable cellspositive for CD11b or CD14 surface expression.

Pregnadienediones Induce Apoptosis in AML1984




ResultsGuggulsterone Isomers and 16-Dehydroprogesterone

Prevent the Growth of Leukemic Cells in Culture byInducing ApoptosisBecause there are no reports in the literature investigat-

ing the antiproliferative effects of guggulsterone or relatedpregnadienediones, we cultured HL60 and U937 cells withincreasing concentrations of both guggulsterone isomersand 16-dehydroprogesterone for 72 and 144 hours andquantitated the number of viable cells remaining aftertreatment. The results in Fig. 1B show that cis-guggul-sterone, trans-guggulsterone, and 16-dehydroprogesteroneinhibited the proliferation of HL60 cells in a time- anddose-dependent manner displaying 144-hour IC50s rang-ing from 8.3 to 10.9 Amol/L. Similarly, all threecompounds inhibited the proliferation of U937 cells withslightly higher potencies displaying IC50s varying from 3.6to 8.7 Amol/L (Fig. 1C). To investigate whether apoptosiscontributed to the antiproliferative effects of the guggul-sterone and 16-dehydroprogesterone, we quantitated thepercentage of HL60 and U937 cells that externalized

phosphatidylserine after treatment with these agents for72 hours. We found that all three compounds significantlyincreased phosphatidylserine externalization, albeit cis-guggulsterone was the most potent compound displaying72-hour IC50s of 16.1 and 19.8 Amol/L after treatment ofHL60 and U937 cells, respectively, for 72 hours (Fig. 2A).Furthermore, the increase in phosphatidylserine external-ization correlated with a marked loss in mitochondrialmembrane potential (DCm) as evidenced by reducedaccumulation of the potentiometric probe TMRM,which was more pronounced in cells treated with cis-guggulsterone (Fig. 2B). Taken together, these resultsshow that the isomeric pregnadienedione steroids guggul-sterone and 16-dehydroprogesterone prevent the prolifer-ation of leukemic cells in culture partly by inducingmitochondrial dysfunction and apoptosis.

Guggulsterone Isomers and 16-DehydroprogesteroneInduce Differentiation of HL60 and NB4Cells in CultureHL60 cells have been shown to differentiate in culture

after treatment with several anticancer drugs and apoptosisinducers with lipophilic and/or steroidal structure, and

Figure 2. Guggulsterone isomers and 16-dehydroprogesterone induce apoptosis in HL60 and U937 cells and promote differentiation of HL60 andNB4 cells in long-term culture. A, HL60 cells were cultured in the presence of increasing concentrations of the guggulsterone isomers and16-dehydroprogesterone (10–20 Amol/L) for 72 h. Phosphatidylserine externalization and DCm were quantitated as described in Materials and Methods. B,U937 cells were treated with the guggulsterone isomers and 16-dehydroprogesterone as for HL60 cells above. C, HL60 cells were treated with 10 Amol/Lcis -guggulsterone, trans -guggulsterone, or 16-dehydroprogesterone for 120 h and the number of cells expressing surface CD11b and CD14 was evaluatedby flow cytometry as described in Materials and Methods. D, NB4 cells were treated with 10 Amol/L cis -guggulsterone, trans -guggulsterone, or 16-dehydroprogesterone for 96 h and the number of cells expressing surface CD14 was evaluated by flow cytometry as above. All experiments were done induplicate and repeated at least thrice. Columns, mean of three independent experiments; bars, SE. *, P < 0.05; **, P < 0.005.





these include the synthetic triterpenoid CDDO-Me, 12-O-tetradecanoylphorbol-13-acetate, oxysterols, and 1,25-dihy-droxyvitamin D3 (25–30). Because one mechanism thatmay contribute to the antiproliferative effects of theguggulsterone and 16-dehydroprogesterone is the induc-tion of differentiation, we examined the surface expressionof the monocytic and myelomonocytic markers CD14 andCD11b, respectively, in HL60 cells treated with 10 Amol/L16-dehydroprogesterone, cis-guggulsterone, and trans-gug-gulsterone for 120 hours. Of note, because the selectivekilling of immature cells by these steroids would increasethe relative numbers of differentiated cells, we calculatedthe absolute number of cells expressing surface CD14 orCD11b as described in Materials and Methods. Theseresults presented in Fig. 2C illustrate that trans-guggulster-one was the more potent inducer of myelomonocyticdifferentiation, promoting a 2.5-fold increase in HL60 cellsexpressing surface CD11b (P < 0.004) followed by 16-dehydroprogesterone, which induced a modest but notstatistically significant 1.7-fold increase (P > 0.05). trans-Guggulsterone also significantly increased the number of

HL60 cells expressing surface CD14 (2.2-fold; P < 0.03),whereas neither 16-dehydroprogesterone nor cis-guggul-sterone promoted an increase in cells expressing thismonocytic marker. Interestingly, cis-guggulsterone seemedto decrease the numbers of cells expressing both surfacemarkers probably owing to its higher cytotoxicity at10 Amol/L compared with 16-dehydroprogesterone ortrans-guggulsterone. Finally, we also investigated if thesesteroids would promote differentiation in a different cellcontext. For these experiments, we treated the acutepromyelocytic leukemia cell line NB4, which has beenshown to undergo monocytic differentiation after treatmentwith retinoic acid and 1,25-dihydroxyvitamin D3 (28–30),with 10 Amol/L guggulsterone and 16-dehydroprogester-one for 96 hours and examined the cell surface expression ofCD14 (Fig. 2D). 16-Dehydroprogesterone and trans-guggul-sterone induced a robust increase in NB4 cells expressingsurface CD14 (3.8- and 3.5-fold, respectively; P < 0.008),whereas cis-guggulsterone induced a more modest butsignificant 1.7-fold increase (P < 0.03). Under theseconditions, 16-dehydroprogesterone and trans-guggulster-one decrease the number of viable NB4 cells by f50%,whereas cis-guggulsterone induced a more pronouncedf80% decrease (data not shown). Notably, we haveobserved that lower concentrations of cis-guggulsterone(<10 Amol/L), which are not markedly cytotoxic, do notinduce an increase in differentiated HL60 or NB4 cells (datanot shown), suggesting that the mild differentiating effectsof this agent are closely associated with its cytotoxicity.Together, our findings indicate that all three steroids canpromote differentiation of AML cell lines in culture, albeitthe higher cytotoxicity of cis-guggulsterone seems to maskthis effect.

Guggulsterone Isomers and 16-DehydroprogesteroneIncrease Generation of Reactive Oxygen Species,Decrease the Phosphorylation of ERK, and Inducethe Expression of Heme Oxygenase-1 in U937 Cells inCultureBecause reactive oxygen species (ROS) have been

implicated in triggering apoptosis, we investigated theeffects of 15 Amol/L of the guggulsterone isomers and 16-dehydroprogesterone on the levels of superoxide radicalsin U937 cells after treatment for 48 hours. The results showthat cis-guggulsterone and trans-guggulsterone induced asignificant 16% and 19% increase in levels of superoxideradicals (O2

�), respectively, compared with cells treatedwith DMSO (P < 0.0001), whereas 16-dehydroprogesteronepromoted a weaker albeit significant (P < 0.0001) 8%increase in O2

� (Fig. 3A). Under these conditions, there wasminimal apoptosis (data not shown), suggesting that theobserved increase in O2

� was not a consequence of celldeath. Previous reports have shown that induction ofapoptosis in U937 cells by agents, such as CDDO-Me,bortezomib, adaphostin, and arsenic trioxide, which induceoxidative stress, is accompanied by inhibition of ERK phos-phorylation (31–34). We therefore investigated if the effectof guggulsterone isomers and 16-dehydroprogesteroneon the levels of phosphorylated ERK (pERK) in U937 cells

Figure 3. Guggulsterone isomers and 16-dehydroprogesterone inducedthe generation of O2

�, decrease the activation of ERK, and induceexpression of the stress response protein heme oxygenase-1. A, U937cells were treated with 20 Amol/L cis -guggulsterone, trans -guggulsterone,and 16-dehydroprogesterone for 24 h and the levels of O2

� werequantitated by flow cytometry as described in Materials and Methods.B, U937 cells were treated with 15 Amol/L cis -guggulsterone, trans -guggulsterone, or 16-dehydroprogesterone for 48 h and the levels ofpERK, total ERK, heme oxygenase-1 (HO-1 ), and a-tubulin were examinedby Western blot as described in Materials and Methods. Superoxidemeasurements were done in triplicate and repeated at least twice.Columns, mean of a representative experiment; bars, SD. *, P < 0.05;**, P < 0.0005.





by Western blot. Guggulsterone isomers induced af90% decrease in the levels of pERK after 48 hourscompared with cells treated with vehicle (DMSO). Incontrast, U937 cells treated with 16-dehydroprogesteroneonly exhibited a f 20% inhibition of ERK phosphorylation.In addition, the enhanced formation of O2

� promoted by theguggulsterone isomers and 16-dehydroprogesterone wasassociated with increased expression of the oxidative stressresponsive gene heme oxygenase-1 (Fig. 3B), and thisincrease was greater in cells treated with cis-guggulsterone(38-fold) and trans-guggulsterone (32-fold) than in cellstreated with 16-dehydroprogesterone (3-fold). Thus, gug-gulsterone isomers and, to a lesser extent, 16-dehydropro-gesterone induce oxidative stress in U937 cells that isaccompanied by decreased activation of ERK and increasedlevels of heme oxygenase-1.

Rapid Cytotoxicity of Higher Concentrations ofGuggulsterone Isomers and16-Dehydroprogesterone IsAssociated with ROSGeneration, Inactivation of ERK,and InductionofApoptosisTo further investigate the short-term cytotoxic effects of

the guggulsterone isomers and 16-dehydroprogesterone,we treated U937 cells with higher concentrations of theseagents (25–75 Amol/L) for 20 hours. Our results show that,as observed for lower concentrations (10–20 Amol/L) ofguggulsterone isomers and 16-dehydroprogesterone, higherconcentrations of these agents also induced a dose-dependent externalization of phosphatidylserine, loss ofDCm, and increased generation of O2

� (Fig. 4A), suggestinga priori that at higher concentrations these compounds elicitsimilar cytotoxic responses albeit displaying faster kinetics.Moreover, 16-dehydroprogesterone was as effective asguggulsterone isomers in decreasing the levels of pERKafter treatment with 50 Amol/L for 20 hours and thiscorrelates with its comparable ability to induce ROS at theseconcentrations. The 50 Amol/L concentrations of all com-pounds induced heme oxygenase-1 expression and cleavageof caspase-3, suggesting that the observed oxidative stresscorrelates with caspase activation. To further investigate thekinetics of oxidative stress induced by 16-dehydroprogester-one and the guggulsterone, we quantitated the generation ofO2

� in cells treated with these compounds for 3 and 6 hours.Interestingly, at 3 hours, 16-dehydroprogesterone and trans-guggulsterone induced a significant (P < 0.05) accumulationof O2

� (6.6- and 5.3-fold, respectively), whereas cis-guggul-sterone failed to significantly increase the levels ofO2

� (P > 0.05; Fig. 4B). Increased generation of ROS by 16-dehydroprogesterone and trans-guggulsteronewas sustainedfor 6 hours, and at this time, cis-guggulsterone showed asignificant (P < 0.003) 7.8-fold increase in the O2

� levels,suggesting that, although all three agents provoke ROSgeneration, they induce this response with different kinetics.

Cytotoxic Concentrations of the Guggulsterone Iso-mers and16-Dehydroprogesterone Uncover a SelectiveDepletion of Reduced Glutathione and Oxidation ofCardiolipin Inducedby cis-GuggulsteroneBecause reduced glutathione levels are critical determi-

nants of intracellular redox homeostasis (35), we also

investigated the effects of guggulsterone isomers and 16-dehydroprogesterone on the levels of this intracellularantioxidant in U937 cells. Interestingly, at 3 hours, cis-guggulsterone induced a significant (P < 0.0002) 24%decrease in the levels of intracellular glutathione, whereastrans-guggulsterone, which induced significant increasesin ROS at this time point, failed to affect the levels ofglutathione; U937 cells treated with 16-dehydroprogester-one for 3 hours only displayed a slight albeit significant(P < 0.003) 7% decrease in glutathione (Fig. 4C). Thedecrease in glutathione induced by cis-guggulsterone wasmaintained 6 hours after treatment, and at this time, neither16-dehydroprogesterone nor trans-guggulsterone elicited asignificant decrease (P > 0.05) in the levels of this anti-oxidant in U937 cells. Because cis-guggulsterone decreasedthe levels of intracellular glutathione, we investigated ifthis pregnadienedione would promote oxidation of themitochondrial phospholipid cardiolipin. Cardiolipin isessential for mitochondrial function and for preventingapoptosis by sequestering cytochrome c (36, 37). Glutathi-one is required for maintaining appropriate levels ofreduced cardiolipin via the action of a glutathione-dependent peroxidase that is antiapoptotic (38). Indeed,consistent with the effects of cis-guggulsterone on gluta-thione, the results presented in Fig. 4D show that after20 hours treatment cis-guggulsterone induced a dramaticincrease in the percentage of U937 cells with low levels ofcardiolipin, whereas 16-dehydroprogesterone and trans-guggulsterone failed to elicit a similar response. Takentogether, these results show that, although all three pregna-dienediones induce rapid generation of ROS in U937 cells,they display different kinetics and only cis-guggulsteroneprovokes marked decreases in glutathione and substantialloss of cardiolipin.

Mitochondrial Dysfunction Induced by the Guggul-sterone Isomers and 16-Dehydroprogesterone Is Inde-pendent of CaspaseActivationBecause it has been reported recently that caspases

mediate loss of DCm induced by a variety of proapoptoticstimuli (39, 40), we investigated if mitochondrial dysfunc-tion and apoptosis induced by 50 Amol/L guggulsteroneand 16-dehydroprogesterone after 24 hours was dependenton the activity of these proteases. In addition, we alsoinvestigated if the potent antioxidant N-acetylcysteinecould prevent cytotoxicity induced by guggulsterone and16-dehydroprogesterone. The results presented in Fig. 5Ashow that loss of DCm induced by all three compounds wasnot affected by pharmacologic inhibition of caspases usingthe pancaspase inhibitor z-VAD-fmk. In contrast, z-VAD-fmk significantly (P < 0.001) prevented the externalizationof phosphatidylserine induced by all three compounds,suggesting that caspase inhibition in cells treated withguggulsterone and 16-dehydroprogesterone switches themode of cell death from apoptosis to necrosis (Fig. 5B).Interestingly, the antioxidant N-acetylcysteine completelyprevented the cytotoxicity induced by 16-dehydroproges-terone but not that induced by cis-guggulsterone or trans-guggulsterone, suggesting that 16-dehydroprogesterone





may depend solely on ROS to induce cell death. Thesedata illustrate that the cis-guggulsterone and trans-guggulsterone, but not 16-dehydroprogesterone, inducecytotoxicity independent of the generation of ROS andthat all three compounds induce mitochondrial dysfunc-tion in the absence of caspase activation, but caspasescontribute to the onset of apoptosis in cells treated withthese agents.

Guggulsterone Isomers and 16-DehydroprogesteroneInduce Mitochondrial Dysfunction and Apoptosis inCD34-Positive Cells from Primary Leukemia SamplesTo determine if the guggulsterone and 16-dehydropro-

gesterone would induce apoptosis in CD34-positive cellsfrom primary leukemia samples, we exposed ex vivo fourprimary leukemic samples to increasing concentrations(25–100 Amol/L) of these agents for 15 hours andexamined externalization of phosphatidylserine in CD34-positive cells by flow cytometry. For three samples, we also

investigated the loss of DCm in CD34-positive cells. Ourresults illustrated in Fig. 6A show that 25 Amol/Lguggulsterone isomers and 16-dehydroprogesterone in-duced phosphatidylserine externalization in CD34-positiveblasts from all samples tested (P < 0.02), albeit sample 3was markedly more resistant to the cytotoxicity of all threecompounds. Furthermore, all three compounds inducedsignificant (P < 0.03) decreases in DCm in CD34-positiveblasts from patients 2 to 4 (Fig. 6B). Finally, we investigatedthe cytotoxicity of 100 Amol/L guggulsterone and16-dehydroprogesterone in normal PBMCs obtained fromhealthy volunteers. As shown in Fig. 6C, none of the threeagents induced apoptosis in normal PBMC to the sameextent as in leukemia blasts. Notably, trans-guggulsteronewas the least cytotoxic pregnadienedione to PBMC mini-mally increasing phosphatidylserine externalization aboveDMSO-treated cells by an average of 2.1 F 5.4% comparedwith 50.7 F 21.6% in leukemia blasts (P < 0.02). Similarly,

Figure 4. Cytotoxicity of higher concentrations ofthe guggulsterone isomers and 16-dehydroprogesteroneis still mediated by the generation of O2

� and theinduction of apoptosis but differentiates cis -guggulster-one from trans -guggulsterone and 16-dehydroproges-terone. A, U937 cells were treated with increasingconcentrations of theguggulsterone isomers and16-dehydroprogesterone (25–75 Amol/L) for 20 h, andphosphatidylserine externalization, DCm, and O2

�

generation were quantitated as described in Materialsand Methods. In addition, the levels of pERK,total ERK, heme oxygenase-1, and a-tubulin wereexamined. B, U937 cells were treated with 75 Amol/Lcis -guggulsterone, trans -guggulsterone, and 16-dehydroprogesterone for 3 and 6 h and O2

� generationwas quantitated as above. C, U937 cells weretreated with 75 Amol/L cis -guggulsterone, trans -guggulsterone, and 16-dehydroprogesterone for 3and 6 h and intracellular glutathione was quantitatedas described in Materials and Methods. D, U937cells were treated with increasing concentrationsof the guggulsterone and 16-dehydroprogesterone(25–75 Amol/L) for 20 h and the oxidation of cardiolipinwas examined by flow cytometry as described inMaterials and Methods. Flow cytometry experimentswere done in triplicate and repeated at least twice.Points, mean of a representative experiment; bars, SD.*, P < 0.05; **, P < 0.0005.





16-dehydroprogesterone induced significantly less apopto-sis in normal PBMC than in leukemia blasts (11.5 F 6%versus 47.9 F 20.7%; P < 0.04). cis-Guggulsterone exhibitedthe highest cytotoxicity in PBMC increasing phosphatidyl-serine externalization by 22.5 F 7.7%, albeit this increasewas significantly lower (P < 0.05) than the observed 60.7 F23.5% increase in leukemia blasts treated with this agent.Taken together, these findings show that guggulsteroneisomers and 16-dehydroprogesterone effectively induceapoptosis in CD34-positive cells from primary leukemiasamples but not in normal PBMC and that apoptosisinduced in leukemia blasts is associated with markedmitochondrial dysfunction.

DiscussionNatural products have provided a large number ofcurrently used chemotherapeutics and will continue to be

an important component of drug discovery (41, 42). In fact,there are >1,000 species of plants that possess anticancerproperties (43), and many active biological componentshave been chemically modified to generate promising newchemotherapeutic drugs (44–46). We have shown previ-ously that synthetic derivatives of oleanolic acid anddiindolylmethane, found in the oleander tree and crucifer-ous vegetables, respectively, potently induced apoptosisin leukemic cell lines and primary leukemic samples(26, 47, 48). The naturally occurring plant sterols, theguggulsterones, are the active components of the antilipi-demic extract of the guggul tree C. mukul that are currentlybeing evaluated for treatment of hypercholesterolemia andobesity (8, 10, 11). Interestingly, these pregnane sterols alsopossess anti-inflammatory activity that may be in partdependent on their ability to inhibit nuclear factor-nBsignaling (15–17, 23). We thus hypothesized a priori thatlike other nuclear factor-nB inhibitors, such as curcuminand betulinic acid, the guggulsterone isomers would alsodisplay antiproliferative activities against leukemic cellsin culture.We first investigated the effects of both cis-guggulster-

one and trans-guggulsterone isomers as well as 16-dehydroprogesterone, a steroidal isomer of guggulsterone,on the long-term proliferation of U937 and HL60 cells inculture. Our results show that the guggulsterone isomersas well as 16-dehydroprogesterone similarly inhibited theproliferation of both cell lines in culture, suggesting thatthe pregnadienedione scaffold of these agents may be animportant structural feature required for their antiproli-ferative activity. In addition, our results indicate thatapoptosis contributes, at least in part, to the antiprolifer-ative effects of all three compounds, and this isassociated with marked mitochondrial dysfunction inboth cell lines. We also investigated the ability of theguggulsterone isomers and 16-dehydroprogesterone toinduce expression of differentiation markers on thesurface of HL60 cells and found that at a concentrationof 10 Amol/L trans-guggulsterone, but not cis-guggul-sterone or 16-dehydroprogesterone, induced a significantincrease in the number of cells expressing CD11b andCD14 after treatment for 120 hours, suggesting thatmyelomonocytic and monocytic differentiation contributeto the antiproliferative effects of trans-guggulsterone inHL60 cells. Of note, because cis-guggulsterone wasobserved to be more cytotoxic than trans-guggulsteroneor 16-dehydroprogesterone under these conditions, wehypothesize that any differentiating effects of cis-guggul-sterone in HL60 cells may be masked by its increasedcytotoxicity. Further investigation of the differentiatingactivity of these steroids revealed that all three com-pounds significantly increased the number of NB4 cellsexpressing CD14, suggesting that these steroids canpromote monocytic differentiation in a cell context–dependent manner. These are the first data that showthe antiproliferative, proapoptotic, and differentiatingeffects of the guggulsterone isomers and 16-dehydropro-gesterone in leukemic cells in culture.

Figure 5. Mitochondrial dysfunction induced by the guggulsteroneisomers and 16-dehydroprogesterone is independent of caspase activationand the antioxidant N -acetylcysteine (NAC ) only prevents cell deathinduced by 16-dehydroprogesterone but not by cis -guggulsterone ortrans -guggulsterone. Briefly, U937 cells were treated with 50 Amol/L cis -guggulsterone, trans -guggulsterone, or 16-dehydroprogesterone alone orin combination with the pancaspase inhibitor z-VAD-fmk (50 Amol/L) orthe potent antioxidant N -acetylcysteine (5 mmol/L), and DCm (A) andphosphatidylserine externalization (B) were quantitated after 24 h.





Figure 6. Guggulsterone isomers and 16-dehydroprogesteroneinduced mitochondrial dysfunction and apoptosis in CD34-positivecells from primary leukemic samples. Briefly, patient samples werecollected as described in Materials and Methods and cultured in thepresence of increasing concentrations (25–100 Amol/L) of cis -guggulsterone, trans -guggulsterone, or 16-dehydroprogesteronefor 15 h. Phosphatidylserine externalization (A) and DCm (B) werequantitated in CD34-positive cells. C, PBMC samples from threehealthy volunteers (A–C) were exposed to 100 Amol/L guggul-sterone and 16-dehydroprogesterone for 20 h and phosphatidyl-serine externalization was quantitated by flow cytometry.





Because the generation of O2� is an early event in many

forms of cell death and is an indicator of mitochondrialdysfunction (49, 50), we also examined if the cytotoxicityof low concentrations (<20 Amol/L) of the guggulsteroneisomers and 16-dehydroprogesterone was associated withincreased generation of this ROS. We observed that theguggulsterone isomers and, to a lesser extent, 16-dehydro-progesterone indeed generated increased levels of O2

�

before the onset of apoptosis, suggesting that the long-termcytotoxicity of these agents is associated with oxidativestress. Interestingly, our observations also indicate that atlow concentrations (<20 Amol/L) cis-guggulsterone andtrans -guggulsterone, but not 16-dehydroprogesterone,markedly decreased the pERK expression after treatmentfor 48 hours, suggesting that the increased ROS generatedby these compounds may contribute to the inactivation ofERK signaling (32, 33). Finally, we observed that cytotox-icity induced by cis-guggulsterone, trans-guggulsterone,and 16-dehydroprogesterone was accompanied by in-creased expression of the oxidative stress response proteinheme oxygenase-1, and cis -guggulsterone and trans -guggulsterone were more effective than 16-dehydropro-gesterone in inducing this response probably due to theirincreased ability to generate ROS. These data indicatethat oxidative stress is associated with the cytotoxicityof the guggulsterone and 16-dehydroprogesterone andthat the guggulsterone can abrogate the activation of ERKin leukemic cells.To further investigate the mechanism of action of the

guggulsterone isomers and 16-dehydroprogesterone, weevaluated the short-term effects of higher concentrations(25–75 Amol/L) of these agents. Our results indicate that athigher concentrations the cytotoxicity of these agents is stillassociated with apoptosis, mitochondrial dysfunction, andROS generation. In addition, at higher concentrations, 16-dehydroprogesterone was as effective as the guggulsteroneisomers in decreasing pERK levels and this correlated withits increased ability to induce ROS at these concentrations.However, although all three agents induced a dose- andtime-dependent increase in the generation of ROS, only cis-guggulsterone significantly decreased glutathione levels,and this occurred before the increase in O2

� levels,suggesting that cis-guggulsterone may act through adifferent mechanism to induce oxidative stress. Notably,only cis-guggulsterone markedly decreased the levels ofcardiolipin, suggesting that the decrease in glutathioneinduced by this agent may lead to oxidation of this criticalmitochondrial phospholipid. The use of higher concen-trations of the guggulsterone and 16-dehydroprogesteroneuncovered a cis-guggulsterone-specific effect on the levelsof glutathione and cardiolipin in U937 cells, suggesting thatthe cytotoxicity of cis-guggulsterone may be mediated by adifferent mechanism from that of trans-guggulsterone or16-dehydroprogesterone.We also investigated if caspases were involved in the

cytotoxicity of the guggulsterone and 16-dehydroproges-terone and found that pharmacologic inhibition of theseproteases with z-VAD-fmk prevented phosphatidylserine

externalization but not loss of DCm, suggesting thatmitochondrial dysfunction induced by these agents occursbefore caspase activation but caspases contribute to theinduction of apoptosis. The potent antioxidant N-acetyl-cysteine completely prevented the cytotoxicity of 16-dehydroprogesterone but not that of the guggulsteroneisomers. This observation suggests that either (a) theoxidative stress induced by the guggulsterone cannot bereversed by 5 mmol/L N-acetylcysteine cotreatment or (b)the cytotoxicity of 16-dehydroprogesterone depends solelyon the generation of ROS.Finally, we investigated if the guggulsterone and 16-

dehydroprogesterone could induce apoptosis and mito-chondrial dysfunction in primary CD34-positive leukemiacells in culture. Our results show that all three agentsinduced rapid (f15 hours) apoptosis in CD34-positive cellsfrom primary leukemia samples and that this wasassociated with mitochondrial dysfunction, although onesample seemed to be more resistant to the cytotoxic effectsof these compounds. Most notably, the guggulsterone and16-dehydroprogesterone were more cytotoxic to leukemiablasts than to normal PBMC, suggesting that the pregna-dienedione structure of these agents may display atherapeutic window. These results are the first to showthe antileukemic activity of the guggulsterone isomers and16-dehydroprogesterone in CD34-positive primary leuke-mic cells.In conclusion, the guggulsterone isomers and 16-dehy-

droprogesterone represent a novel class of naturallyoccurring compounds that exhibit antileukemic activityby inducing apoptosis and differentiation. Our resultsindicate that the pregnadienedione structure of thesesteroid isomers has inherent antiproliferative, proapoptotic,and differentiating activities and may display someselectivity for leukemia blasts over normal PBMC. We arecurrently investigating the antileukemic effect of syntheticderivatives of these agents as well as their pharmacokineticproperties in animal models with the goal of developingnovel and more effective treatments for AML. Theunderstanding of the mechanism of action of this novelclass of steroidal compounds will offer additional targetsfor the treatment of human leukemias.

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2005;4:1982-1992. Mol Cancer Ther Ismael Samudio, Marina Konopleva, Stephen Safe, et al. antileukemic activities of the pregnadienedione structureacute myeloid leukemia: identification of isomer-specific Guggulsterones induce apoptosis and differentiation in

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Guggulsterones induce apoptosis and differentiation in ... · suggest that in addition to its lipid-lowering activity guggulsterone may modulate anti-inflammatory and anti-oxidant