Guggulsterones Induce Apoptosis And

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Guggulsterones induce apoptosis and differentiation in acute myeloid leukemia: identification of isomer-specific antileukemic activities of the pregnadienedione structure

Ismael Samudio,1 Marina Konopleva,1 phosphatidylserine externalization of CD34-positive Stephen Safe,2,3 Teresa McQueen,1 blasts from primary leukemic samples. This study is the and Michael Andreeff1 first to show that guggulsterones and 16-dehydroprogesterone exert antileukemic effects via the induction of 1Section of Molecular Hematology and Therapy, Department of apoptosis and differentiation and, more importantly, Blood and Marrow Transplantation, The University of Texas identifies the pregnadienedione structure as a potential M.D. Anderson Cancer Center; 2Institute of Biosciences and Technology, Texas A&M University, Houston, Texas and chemotherapeutic scaffold. [Mol Cancer Ther 2005; 3Department of Veterinary Physiology and Pharmacology, Texas 4(12):1982–92] A&M University, College Station, Texas Introduction Abstract Acute myeloid leukemias (AML) are clonal malignancies In this study, the antileukemic effects of three isomeric characterized by increased numbers of immature myeloid pregnadienedione steroids [i.e., cis-guggulsterone, progenitor cells arrested at different stages of granulocytic trans-guggulsterone, and 16-dehydroprogesterone] and monocytic differentiation. First-line treatment of AML were investigated in HL60 and U937 cells as well as consists of a combination of cytarabine and an anthracy- in primary leukemic blasts in culture. Our results show cline, and although this combination results in 60% to 80% that all three compounds inhibited the proliferation of complete remissions in newly diagnosed patients, most HL60 and U937 cells, with IC50s ranging from 3.6 to patients will relapse with resistant disease (1). Because 10.9 Mmol/L after treatment for 6 days. These growth achievement of complete remission is a prerequisite for inhibitory effects correlated with externalization of long-term survival (2), several novel therapeutic modalities phosphatidylserine and loss of mitochondrial membrane have been investigated, including the use of different potential, suggesting that these isomeric steroids induce anthracycline formulations, different nucleoside analogues, apoptosis in leukemia cells. z-VAD-fmk prevented phos- and the combination of the antiangiogenic agent thalido- phatidylserine externalization but not mitochondrial mide with cytarabine/anthracycline or topotecan/anthra- membrane potential loss, indicating that mitochondrial cycline (3–5). However, overall improvement in survival dysfunction occurred in the absence of caspase acti- rates has been marginal at best underlining the need for vation. Interestingly, although all three compounds development of more effective therapies. The most striking increased the generation of reactive oxygen species increase of complete remission and survival has been and decreased phosphorylation of extracellular signal- achieved by ligation of the nuclear retinoic acid receptor a regulated kinase, only cis-guggulsterone induced a rapid in acute promyelocytic leukemias with all-trans retinoic depletion of reduced glutathione levels and oxidation of acid (6, 7). the mitochondrial phospholipid cardiolipin. 16-Dehydro- The gum resin from the guggul tree Commiphora mukul progesterone and trans-guggulsterone induced differen- has been used in Ayurvedic medicine for centuries to treat tiation of HL60 and NB4 cells as evidenced by increased inflammatory and lipid disorders (8), and an ethylacetate surface expression of CD11b and/or CD14, and all three extract of the resin, termed guggulipid, has been reported steroids rapidly induced mitochondrial dysfunction and to have an antiobesity and antilipidemic effect in clinical trials with no significant toxicity (9–12). The active substances in guggulipid are the pregnane plant sterols cis-guggulsterone and trans-guggulsterone, which have Received 7/18/05; revised 9/14/05; accepted 9/23/05. been shown to lower cholesterol and triglycerides in The costs of publication of this article were defrayed in part by the normal and high-fat-fed rats (9). The antilipidemic effects payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to of guggulsterone may be mediated by antagonism of the indicate this fact. orphan receptor FXR (13) as well as promiscuous inter- Requests for reprints: Michael Andreeff, Section of Molecular Hematology actions with other nuclear receptors (14). Notably, although and Therapy, Department of Blood and Marrow Transplantation, The most studies on guggulsterone have focused on their University of Texas M.D. Anderson Cancer Center, Unit 448, 1400 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-7260; antilipidemic activity, these compounds have also shown Fax 713-794-474. E-mail: [email protected] potent anti-inflammatory effects, such as preventing Copyright C 2005 American Association for Cancer Research. oxidative damage during isoproterenol-induced myocar- doi:10.1158/1535-7163.MCT-05-0247 dial necrosis in rats (15, 16) and decreasing inflammation

Mol Cancer Ther 2005;4(12). December 2005

associated with nodulocystic acne (17). These observations guggulsterone, like other anti-inflammatory and chemo- suggest that in addition to its lipid-lowering activity preventive agents, may decrease the proliferation of guggulsterone may modulate anti-inflammatory and anticancer cells in culture. Here, we report that both isomers oxidant responses. of guggulsterone, cis-guggulsterone and trans-guggul- A variety of naturally occurring compounds exhibit sterone, effectively inhibit the proliferation of leukemic chemopreventive and anti-inflammatory effects, includ- cancer cell lines and induce apoptosis and differentia- ing resveratrol, betulinic acid, saikosaponin, and curcu- tion. Interestingly, a mammalian steroid metabolite and min. Some of the chemotherapeutic activities of these chemical isomer of guggulsterone, 16-dehydroprogester- compounds may be related to their inhibition of nuclear one, also induced a comparable pattern of differentiation, factor-nB signaling (18–22), and a recent study reported growth inhibition, and apoptosis, suggesting that the that cis-guggulsterone inhibitedtumornecrosisfactor-a– pregnadienedione structure of these steroids (Fig. 1A) induced nuclear factor-nB signaling and sensitized offers the potential for development of novel chemo- cancer cells to apoptosis induced by taxol, doxorubicin, therapeutics. Our results are the first to show the and tumor necrosis factor-a (23). Surprisingly, there are antileukemic effects of guggulsterone isomers and 16- no studies to date investigating the direct antiprolifer- dehydroprogesterone, and current studies are investigat- ative and proapoptotic effects of guggulsterone in cancer ing their mechanism of action and development of more cell lines in culture. We therefore hypothesized that potent 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 the pregnadienedione isomers used in this study. B, HL60 cells were cultured in the presence of increasing concentrations of the guggulsterone isomers and 16-dehydroprogesterone (10 – 20 Amol/L) for 72 and 144 h. Viable cells were counted using a hemocytometer after trypan blue staining. C, U937 cells were treated with the guggulsterone isomers and 16-dehydroprogesterone as for HL60 cells above. All experiments were done in duplicate and repeated at least thrice. cGS, cis-guggulsterone; tGS, trans-guggulsterone; P, 16-dehydroprogesterone. Points, mean of three independent experiments; bars, SE.

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Materials and Methods Measurement of Phosphatidylserine Externalization Cell Lines, Chemicals, and Biochemicals and Mitochondrial Membrane Potential U937 and HL60 cells were maintained in RPMI supple- After appropriate treatments, cells were washed twice in mented with 10% FCS, 1% glutamine, and 100 units/mL PBS and then resuspended in 100 AL Annexin binding j penicillin in a 37 C incubator containing 5% CO2. 16- buffer [140 mmol/L NaCl, 10 mmol/L KH2PO4, 5 mmol/L Dehydroprogesterone, cis-guggulsterone, and trans-gug- CaCl2 (pH 7.4)] containing 25 nmol/L TMRM and 1:100 gulsterone were purchased from Steraloids, Inc. (Newport, dilution of Annexin V-FLUOS (Roche Diagnostics, j RI). TMRM, dihydroethidine, and Cell Tracker Green were Mannheim, Germany) incubated at 37 Cfor30minutes. all obtained from Molecular Probes (Eugene, OR). z-VAD- Cells were then analyzed by flow cytometry in a fmk was purchased from Alexis Biochemicals (Axxora FACSCalibur flow cytometer using a 488 nm argon exci- LLC, San Diego, CA). Phospho–extracellular signal-regu- tation laser. lated kinase (ERK) and total ERK antibodies were Measurement of Reactive Oxygen Species Generation purchased from Cell Signaling Technologies, Inc. (Beverly, After appropriate treatments, cells were harvested MA). Heme oxygenase-1 antibody was purchased from BD by centrifugation, washed in PBS, and loaded with the Biosciences (San Jose, CA) and a-tubulin was purchased O2 -sensitive probe dihydroethidine. Cells were incubated at j from Santa Cruz Biotechnology (Santa Cruz, CA). All other 37 C for 10 minutes and washed in PBS, and FL2 fluo- chemicals used were of the highest purity available. rescence was examined by flow cytometry. Results pre- F Human Subjects sented are means SE of three independent experiments. Bone marrow or peripheral blood samples were obtained Western Blot Analysis for in vitro studies from patients with AML. Samples were Cells where harvested by centrifugation, washed twice in collected during routine diagnostic procedures after in- PBS, and resuspended in ice cold lysis buffer [1% Triton formed consent was obtained in accordance with regu- X-100, 45 mmol/L KCl, 10 mmol/L Tris (pH 7.5)] supple- lations and protocols approved by the Institutional Review mented with protease and phosphatase inhibitors and then Board of The University of Texas M. D. Anderson Cancer subjected to SDS-PAGE in 10% or 12% polyacrylamide gels Center (Houston, TX). Mononuclear cells were separated followed by protein transfer to a Hybond-P membrane by Ficoll-Hypaque (Sigma Chemical, St. Louis, MO) density (Amersham Pharmacia Biotech, Little Chalfont, United gradient centrifugation. Patient sample 1 was a bone Kingdom) and immunoblotting. Signals were detected by marrow aspirate containing 85% blasts from an AML-M1 a PhosphorImager (Storm 860, version 4.0, Molecular relapse patient (7del). Patient sample 2 was a bone Dynamics, Sunnyvale, CA). marrow aspirate containing 95% blasts from an AML-M1 Measurement of Cardiolipin Content relapse patient [t(12,17)]. Patient sample 3 was a bone After appropriate treatments, cells were harvested by marrow aspirate containing 97% blasts from an AML-M2 centrifugation, washed once in PBS, and resuspended in relapse patient (normal cytogenetics). Patient sample 4 was PBS containing 10 nmol/L nonyl acridine orange, a a peripheral blood sample containing 92% blasts from an probe that binds with high affinity to reduced but not j AML-M0 relapse patient [7del; t(11,19)]. To investigate oxidized cardiolipin (24). Cells were incubated 37 Cfor the effects of the guggulsterone and 16-dehydroprogester- 30 minutes and FL1 fluorescence was quantitated by one on normal cells, blood samples from three healthy flow cytometry. HL60 and NB4 Cell Differentiation volunteers (A-C) were obtained and peripheral blood A mononuclear cells (PBMC) were separated by Ficoll- HL60 and NB4 cells were treated with 10 mol/L Hypaque density gradient centrifugation. PBMC samples guggulsterone and 16-dehydroprogesterone, and after were then exposed to 100 Amol/L guggulsterone and 16- 120 hours, the cells were collected, washed once in dehydroprogesterone for 20 hours, and phosphatidylserine PBS, and resuspended in PBS containing 1:100 dilution externalization was quantitated by flow cytometry. of CD11b-phycoerythrin and CD14-FITC (both form BD Measurement of Intracellular Glutathione by Flow Biosciences). Cells were incubated at room temperature Cytometry for 15 minutes, washed in PBS, and analyzed by flow 5 cytometry gating on viable cells as determined by Cells (3 10 /mL; 0.5 mL) were treated with compounds forward and side scatter. Cells stained with mouse IgG- as indicated or with 2 mmol/L diethylmaleate for 30 phycoerythrin and mouse IgG-FITC served as negative minutes. Cells were then collected by centrifugation, controls. In parallel, viable cells were counted in a washed in PBS once, and resuspended in 0.2 mL PBS hemocytometer after trypan blue exclusion. The absolute containing 400 Amol/L Cell Tracker Green and incubated at number of differentiated cells was calculated from the 20jC protected from light for 10 minutes. Cells were then equation: washed in PBS several times, and Cell Tracker Green fluorescence was quantitated by flow cytometry. The mean Cell Tracker Green fluorescence from diethylmaleate- ð½Viable cells ð1 10 4Þ ½%CD14 or CD11bþcellsÞ=100% treated samples was considered to be background and subtracted accordingly. All experiments were done in Results were expressed as the total number of viable cells duplicate and repeated at least thrice. positive for CD11b or CD14 surface expression.

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Results phosphatidylserine after treatment with these agents for Guggulsterone Isomers and 16-Dehydroprogesterone 72 hours. We found that all three compounds significantly Prevent the Growth of Leukemic Cells in Culture by increased phosphatidylserine externalization, albeit cis- Inducing Apoptosis guggulsterone was the most potent compound displaying Because there are no reports in the literature investigat- 72-hour IC50s of 16.1 and 19.8 Amol/L after treatment of ing the antiproliferative effects of guggulsterone or related HL60 and U937 cells, respectively, for 72 hours (Fig. 2A). pregnadienediones, we cultured HL60 and U937 cells with Furthermore, the increase in phosphatidylserine external- increasing concentrations of both guggulsterone isomers ization correlated with a marked loss in mitochondrial and 16-dehydroprogesterone for 72 and 144 hours and membrane potential (DCm) as evidenced by reduced quantitated the number of viable cells remaining after accumulation of the potentiometric probe TMRM, treatment. The results in Fig. 1B show that cis-guggul- which was more pronounced in cells treated with cis- sterone, trans-guggulsterone, and 16-dehydroprogesterone guggulsterone (Fig. 2B). Taken together, these results inhibited the proliferation of HL60 cells in a time- and show that the isomeric pregnadienedione steroids guggul- dose-dependent manner displaying 144-hour IC50s rang- sterone and 16-dehydroprogesterone prevent the prolifer- ing from 8.3 to 10.9 Amol/L. Similarly, all three ation of leukemic cells in culture partly by inducing compounds inhibited the proliferation of U937 cells with mitochondrial dysfunction and apoptosis. slightly higher potencies displaying IC50s varying from 3.6 Guggulsterone Isomers and 16-Dehydroprogesterone to 8.7 Amol/L (Fig. 1C). To investigate whether apoptosis Induce Differentiation of HL60 and NB4 Cells in Culture contributed to the antiproliferative effects of the guggul- HL60 cells have been shown to differentiate in culture sterone and 16-dehydroprogesterone, we quantitated the after treatment with several anticancer drugs and apoptosis percentage of HL60 and U937 cells that externalized inducers 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 and NB4 cells in long-term culture. A, HL60 cells were cultured in the presence of increasing concentrations of the guggulsterone isomers and 16-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/L cis-guggulsterone, trans-guggulsterone, or 16-dehydroprogesterone for 120 h and the number of cells expressing surface CD11b and CD14 was evaluated by 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 in duplicate and repeated at least thrice. Columns, mean of three independent experiments; bars, SE. *, P < 0.05; **, P < 0.005.

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these include the synthetic triterpenoid CDDO-Me, 12-O- HL60 cells expressing surface CD14 (2.2-fold; P < 0.03), tetradecanoylphorbol-13-acetate, oxysterols, and 1,25-dihy- whereas neither 16-dehydroprogesterone nor cis-guggul- droxyvitamin D3 (25–30). Because one mechanism that sterone promoted an increase in cells expressing this may contribute to the antiproliferative effects of the monocytic marker. Interestingly, cis-guggulsterone seemed guggulsterone and 16-dehydroprogesterone is the induc- to decrease the numbers of cells expressing both surface tion of differentiation, we examined the surface expression markers probably owing to its higher cytotoxicity at of the monocytic and myelomonocytic markers CD14 and 10 Amol/L compared with 16-dehydroprogesterone or CD11b, respectively, in HL60 cells treated with 10 Amol/L trans-guggulsterone. Finally, we also investigated if these 16-dehydroprogesterone, cis-guggulsterone, and trans-gug- steroids would promote differentiation in a different cell gulsterone for 120 hours. Of note, because the selective context. For these experiments, we treated the acute killing of immature cells by these steroids would increase promyelocytic leukemia cell line NB4, which has been the relative numbers of differentiated cells, we calculated shown to undergo monocytic differentiation after treatment the absolute number of cells expressing surface CD14 or with retinoic acid and 1,25-dihydroxyvitamin D3 (28–30), CD11b as described in Materials and Methods. These with 10 Amol/L guggulsterone and 16-dehydroprogester- results presented in Fig. 2C illustrate that trans-guggulsterone for 96 hours and examined the cell surface expression of one was the more potent inducer of myelomonocytic CD14 (Fig. 2D). 16-Dehydroprogesterone and trans-guggul- differentiation, promoting a 2.5-fold increase in HL60 cells sterone induced a robust increase in NB4 cells expressing expressing surface CD11b (P < 0.004) followed by 16- surface CD14 (3.8- and 3.5-fold, respectively; P < 0.008), dehydroprogesterone, which induced a modest but not whereas cis-guggulsterone induced a more modest but statistically significant 1.7-fold increase (P > 0.05). trans- significant 1.7-fold increase (P < 0.03). Under these Guggulsterone also significantly increased the number of conditions, 16-dehydroprogesterone and trans-guggulsterone decrease the number of viable NB4 cells by f50%, whereas cis-guggulsterone induced a more pronounced f80% decrease (data not shown). Notably, we have observed that lower concentrations of cis-guggulsterone (<10 Amol/L), which are not markedly cytotoxic, do not induce an increase in differentiated HL60 or NB4 cells (data not shown), suggesting that the mild differentiating effects of this agent are closely associated with its cytotoxicity. Together, our findings indicate that all three steroids can promote differentiation of AML cell lines in culture, albeit the higher cytotoxicity of cis-guggulsterone seems to mask this effect. Guggulsterone Isomers and 16-Dehydroprogesterone Increase Generation of Reactive Oxygen Species, Decrease the Phosphorylation of ERK, and Induce the Expression of Heme Oxygenase-1 in U937 Cells in Culture Because reactive oxygen species (ROS) have been implicated in triggering apoptosis, we investigated the effects of 15 Amol/L of the guggulsterone isomers and 16- dehydroprogesterone on the levels of superoxide radicals in U937 cells after treatment for 48 hours. The results show that cis-guggulsterone and trans-guggulsterone induced a significant 16% and 19% increase in levels of superoxide radicals (O2 ), respectively, compared with cells treated with DMSO (P < 0.0001), whereas 16-dehydroprogesterone promoted a weaker albeit significant (P < 0.0001) 8% Figure 3. Guggulsterone isomers and 16-dehydroprogesterone induced increase in O2 (Fig. 3A). Under these conditions, there was the generation of O2, decrease the activation of ERK, and induce expression of the stress response protein heme oxygenase-1. A, U937 minimal apoptosis (data not shown), suggesting that the cells were treated with 20 Amol/L cis-guggulsterone, trans-guggulsterone, observed increase in O2 was not a consequence of cell and 16-dehydroprogesterone for 24 h and the levels of O2 were death. Previous reports have shown that induction of quantitated by flow cytometry as described in Materials and Methods. B, U937 cells were treated with 15 Amol/L cis-guggulsterone, trans- apoptosis in U937 cells by agents, such as CDDO-Me, guggulsterone, or 16-dehydroprogesterone for 48 h and the levels of bortezomib, adaphostin, and arsenic trioxide, which induce pERK, total ERK, heme oxygenase-1 (HO-1), and a-tubulin were examined oxidative stress, is accompanied by inhibition of ERK phos- by Western blot as described in Materials and Methods. Superoxide phorylation (31–34). We therefore investigated if the effect measurements were done in triplicate and repeated at least twice. Columns, mean of a representative experiment; bars, SD. *, P < 0.05; of guggulsterone isomers and 16-dehydroprogesterone **, P < 0.0005. on the levels of phosphorylated ERK (pERK) in U937 cells

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by Western blot. Guggulsterone isomers induced a investigated the effects of guggulsterone isomers and 16- f90% decrease in the levels of pERK after 48 hours dehydroprogesterone on the levels of this intracellular compared with cells treated with vehicle (DMSO). In antioxidant in U937 cells. Interestingly, at 3 hours, cis- contrast, U937 cells treated with 16-dehydroprogesterone guggulsterone induced a significant (P < 0.0002) 24% only exhibited a f 20% inhibition of ERK phosphorylation. decrease in the levels of intracellular glutathione, whereas In addition, the enhanced formation of O2 promoted by the trans-guggulsterone, which induced significant increases guggulsterone isomers and 16-dehydroprogesterone was in ROS at this time point, failed to affect the levels of associated with increased expression of the oxidative stress glutathione; U937 cells treated with 16-dehydroprogester- responsive gene heme oxygenase-1 (Fig. 3B), and this one for 3 hours only displayed a slight albeit significant increase was greater in cells treated with cis-guggulsterone (P < 0.003) 7% decrease in glutathione (Fig. 4C). The (38-fold) and trans-guggulsterone (32-fold) than in cells decrease in glutathione induced by cis-guggulsterone was treated with 16-dehydroprogesterone (3-fold). Thus, gug- maintained 6 hours after treatment, and at this time, neither gulsterone isomers and, to a lesser extent, 16-dehydropro- 16-dehydroprogesterone nor trans-guggulsterone elicited a gesterone induce oxidative stress in U937 cells that is significant decrease (P > 0.05) in the levels of this anti- accompanied by decreased activation of ERK and increased oxidant in U937 cells. Because cis-guggulsterone decreased levels of heme oxygenase-1. the levels of intracellular glutathione, we investigated if Rapid Cytotoxicity of Higher Concentrations of this pregnadienedione would promote oxidation of the Guggulsterone Isomers and 16-Dehydroprogesterone Is mitochondrial phospholipid cardiolipin. Cardiolipin is Associated with ROS Generation, Inactivation of ERK, essential for mitochondrial function and for preventing and Induction of Apoptosis apoptosis by sequestering cytochrome c (36, 37). Glutathi- To further investigate the short-term cytotoxic effects of one is required for maintaining appropriate levels of the guggulsterone isomers and 16-dehydroprogesterone, reduced cardiolipin via the action of a glutathione- we treated U937 cells with higher concentrations of these dependent peroxidase that is antiapoptotic (38). Indeed, agents (25–75 Amol/L) for 20 hours. Our results show that, consistent with the effects of cis-guggulsterone on gluta- as observed for lower concentrations (10–20 Amol/L) of thione, the results presented in Fig. 4D show that after guggulsterone isomers and 16-dehydroprogesterone, higher 20 hours treatment cis-guggulsterone induced a dramatic concentrations of these agents also induced a dose- increase in the percentage of U937 cells with low levels of dependent externalization of phosphatidylserine, loss of cardiolipin, whereas 16-dehydroprogesterone and trans- DCm, and increased generation of O2 (Fig. 4A), suggesting guggulsterone failed to elicit a similar response. Taken a priori that at higher concentrations these compounds elicit together, these results show that, although all three pregna- similar cytotoxic responses albeit displaying faster kinetics. dienediones induce rapid generation of ROS in U937 cells, Moreover, 16-dehydroprogesterone was as effective as they display different kinetics and only cis-guggulsterone guggulsterone isomers in decreasing the levels of pERK provokes marked decreases in glutathione and substantial after treatment with 50 Amol/L for 20 hours and this loss of cardiolipin. correlates with its comparable ability to induce ROS at these Mitochondrial Dysfunction Induced by the Guggul- concentrations. The 50 Amol/L concentrations of all com- sterone Isomers and 16-Dehydroprogesterone Is Inde- pounds induced heme oxygenase-1 expression and cleavage pendent of Caspase Activation of caspase-3, suggesting that the observed oxidative stress Because it has been reported recently that caspases correlates with caspase activation. To further investigate the mediate loss of DCm induced by a variety of proapoptotic kinetics of oxidative stress induced by 16-dehydroprogester- stimuli (39, 40), we investigated if mitochondrial dysfunc- one and the guggulsterone, we quantitated the generation of tion and apoptosis induced by 50 Amol/L guggulsterone O2 in cells treated with these compounds for 3 and 6 hours. and 16-dehydroprogesterone after 24 hours was dependent Interestingly, at 3 hours, 16-dehydroprogesterone and trans- on the activity of these proteases. In addition, we also guggulsterone induced a significant (P < 0.05) accumulation investigated if the potent antioxidant N-acetylcysteine of O2 (6.6- and 5.3-fold, respectively), whereas cis-guggul- could prevent cytotoxicity induced by guggulsterone and sterone failed to significantly increase the levels of 16-dehydroprogesterone. The results presented in Fig. 5A O2 (P > 0.05; Fig. 4B). Increased generation of ROS by 16- show that loss of DCm induced by all three compounds was dehydroprogesterone and trans-guggulsterone was sustained not affected by pharmacologic inhibition of caspases using for 6 hours, and at this time, cis-guggulsterone showed a the pancaspase inhibitor z-VAD-fmk. In contrast, z-VAD- significant (P < 0.003) 7.8-fold increase in the O2 levels, fmk significantly (P < 0.001) prevented the externalization suggesting that, although all three agents provoke ROS of phosphatidylserine induced by all three compounds, generation, they induce this response with different kinetics. suggesting that caspase inhibition in cells treated with Cytotoxic Concentrations of the Guggulsterone Iso- guggulsterone and 16-dehydroprogesterone switches the mers and 16-Dehydroprogesterone Uncover a Selective mode of cell death from apoptosis to necrosis (Fig. 5B). Depletion of Reduced Glutathione and Oxidation of Interestingly, the antioxidant N-acetylcysteine completely Cardiolipin Induced by cis-Guggulsterone prevented the cytotoxicity induced by 16-dehydroproges- Because reduced glutathione levels are critical determi- terone but not that induced by cis-guggulsterone or trans- nants of intracellular redox homeostasis (35), we also guggulsterone, suggesting that 16-dehydroprogesterone

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Figure 4. Cytotoxicity of higher concentrations of the guggulsterone isomers and 16-dehydroprogesterone is still mediated by the generation of O2 and the induction of apoptosis but differentiates cis-guggulsterone from trans-guggulsterone and 16-dehydroprogesterone. A, U937 cells were treated with increasing concentrations of theguggulsterone isomers and 16-dehydroprogesterone (25 – 75 Amol/L) for 20 h, and phosphatidylserine externalization, DCm,andO2 generation were quantitated as described in Materials and Methods. In addition, the levels of pERK, total ERK, heme oxygenase-1, and a-tubulin were examined. B, U937 cells were treated with 75 Amol/L cis-guggulsterone, trans-guggulsterone, and 16- dehydroprogesterone for 3 and 6 h and O2 generation was quantitated as above. C, U937 cells were treated with 75 Amol/L cis-guggulsterone, trans- guggulsterone, and 16-dehydroprogesterone for 3 and 6 h and intracellular glutathione was quantitated asdescribedinMaterialsandMethods.D, U937 cells were treated with increasing concentrations of the guggulsterone and 16-dehydroprogesterone (25 – 75 Amol/L) for 20 h and the oxidation of cardiolipin was examined by flow cytometry as described in Materials and Methods. Flow cytometry experiments were done in triplicate and repeated at least twice. Points, mean of a representative experiment; bars, SD. *, P < 0.05; **, P < 0.0005.

may depend solely on ROS to induce cell death. These investigated the loss of DCm in CD34-positive cells. Our data illustrate that the cis-guggulsterone and trans- results illustrated in Fig. 6A show that 25 Amol/L guggulsterone, but not 16-dehydroprogesterone, induce guggulsterone isomers and 16-dehydroprogesterone in- cytotoxicity independent of the generation of ROS and duced phosphatidylserine externalization in CD34-positive that all three compounds induce mitochondrial dysfunc- blasts from all samples tested (P < 0.02), albeit sample 3 tion in the absence of caspase activation, but caspases was markedly more resistant to the cytotoxicity of all three contribute to the onset of apoptosis in cells treated with compounds. Furthermore, all three compounds induced these agents. significant (P < 0.03) decreases in DCm in CD34-positive Guggulsterone Isomers and 16-Dehydroprogesterone blasts from patients 2 to 4 (Fig. 6B). Finally, we investigated Induce Mitochondrial Dysfunction and Apoptosis in the cytotoxicity of 100 Amol/L guggulsterone and CD34-PositiveCellsfromPrimaryLeukemiaSamples 16-dehydroprogesterone in normal PBMCs obtained from To determine if the guggulsterone and 16-dehydropro- healthy volunteers. As shown in Fig. 6C, none of the three gesterone would induce apoptosis in CD34-positive cells agents induced apoptosis in normal PBMC to the same from primary leukemia samples, we exposed ex vivo four extent as in leukemia blasts. Notably, trans-guggulsterone primary leukemic samples to increasing concentrations was the least cytotoxic pregnadienedione to PBMC mini- (25–100 Amol/L) of these agents for 15 hours and mally increasing phosphatidylserine externalization above examined externalization of phosphatidylserine in CD34- DMSO-treated cells by an average of 2.1 F 5.4% compared positive cells by flow cytometry. For three samples, we also with 50.7 F 21.6% in leukemia blasts (P < 0.02). Similarly,

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16-dehydroprogesterone induced significantly less apopto- an important component of drug discovery (41, 42). In fact, sis in normal PBMC than in leukemia blasts (11.5 F 6% there are >1,000 species of plants that possess anticancer versus 47.9 F 20.7%; P < 0.04). cis-Guggulsterone exhibited properties (43), and many active biological components the highest cytotoxicity in PBMC increasing phosphatidyl- have been chemically modified to generate promising new serine externalization by 22.5 F 7.7%, albeit this increase chemotherapeutic drugs (44–46). We have shown previ- was significantly lower (P < 0.05) than the observed 60.7 F ously that synthetic derivatives of oleanolic acid and 23.5% increase in leukemia blasts treated with this agent. diindolylmethane, found in the oleander tree and crucifer- Taken together, these findings show that guggulsterone ous vegetables, respectively, potently induced apoptosis isomers and 16-dehydroprogesterone effectively induce in leukemic cell lines and primary leukemic samples apoptosis in CD34-positive cells from primary leukemia (26, 47, 48). The naturally occurring plant sterols, the samples but not in normal PBMC and that apoptosis guggulsterones, are the active components of the antilipi- induced in leukemia blasts is associated with marked demic extract of the guggul tree C. mukul that are currently mitochondrial dysfunction. being evaluated for treatment of hypercholesterolemia and obesity (8, 10, 11). Interestingly, these pregnane sterols also Discussion possess anti-inflammatory activity that may be in part dependent on their ability to inhibit nuclear factor-nB Natural products have provided a large number of signaling (15–17, 23). We thus hypothesized a priori that currently used chemotherapeutics and will continue to be like other nuclear factor-nB inhibitors, such as curcumin and betulinic acid, the guggulsterone isomers would also display antiproliferative activities against leukemic cells in culture. We first investigated the effects of both cis-guggulsterone and trans-guggulsterone isomers as well as 16- dehydroprogesterone, a steroidal isomer of guggulsterone, on the long-term proliferation of U937 and HL60 cells in culture. Our results show that the guggulsterone isomers as well as 16-dehydroprogesterone similarly inhibited the proliferation of both cell lines in culture, suggesting that the pregnadienedione scaffold of these agents may be an important structural feature required for their antiproliferative activity. In addition, our results indicate that apoptosis contributes, at least in part, to the antiproliferative effects of all three compounds, and this is associated with marked mitochondrial dysfunction in both cell lines. We also investigated the ability of the guggulsterone isomers and 16-dehydroprogesterone to induce expression of differentiation markers on the surface of HL60 cells and found that at a concentration of 10 Amol/L trans-guggulsterone, but not cis-guggulsterone or 16-dehydroprogesterone, induced a significant increase in the number of cells expressing CD11b and CD14 after treatment for 120 hours, suggesting that myelomonocytic and monocytic differentiation contribute to the antiproliferative effects of trans-guggulsterone in HL60 cells. Of note, because cis-guggulsterone was observed to be more cytotoxic than trans-guggulsterone or 16-dehydroprogesterone under these conditions, we hypothesize that any differentiating effects of cis-guggulsterone in HL60 cells may be masked by its increased cytotoxicity. Further investigation of the differentiating activity of these steroids revealed that all three com- Figure 5. Mitochondrial dysfunction induced by the guggulsterone pounds significantly increased the number of NB4 cells isomers and 16-dehydroprogesterone is independent of caspase activation and the antioxidant N-acetylcysteine (NAC) only prevents cell death expressing CD14, suggesting that these steroids can induced by 16-dehydroprogesterone but not by cis-guggulsterone or promote monocytic differentiation in a cell context– trans-guggulsterone. Briefly, U937 cells were treated with 50 Amol/L cis- dependent manner. These are the first data that show guggulsterone, trans-guggulsterone, or 16-dehydroprogesterone alone or the antiproliferative, proapoptotic, and differentiating in combination with the pancaspase inhibitor z-VAD-fmk (50 Amol/L) or effects of the guggulsterone isomers and 16-dehydropro- the potent antioxidant N-acetylcysteine (5 mmol/L), and DCm (A) and phosphatidylserine externalization (B) were quantitated after 24 h. gesterone in leukemic cells in culture.

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Figure 6. Guggulsterone isomers and 16-dehydroprogesterone induced mitochondrial dysfunction and apoptosis in CD34-positive cells from primary leukemic samples. Briefly, patient samples were collected as described in Materials and Methods and cultured in the presence of increasing concentrations (25 – 100 Amol/L) of cis- guggulsterone, trans-guggulsterone, or 16-dehydroprogesterone for 15 h. Phosphatidylserine externalization (A) and DCm (B) were quantitated in CD34-positive cells. C, PBMC samples from three healthy volunteers (A – C) were exposed to 100 Amol/L guggulsterone and 16-dehydroprogesterone for 20 h and phosphatidylserine externalization was quantitated by flow cytometry.

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Because the generation of O2 is an early event in many externalization but not loss of DCm,suggestingthat forms of cell death and is an indicator of mitochondrial mitochondrial dysfunction induced by these agents occurs dysfunction (49, 50), we also examined if the cytotoxicity before caspase activation but caspases contribute to the of low concentrations (<20 Amol/L) of the guggulsterone induction of apoptosis. The potent antioxidant N-acetyl- isomers and 16-dehydroprogesterone was associated with cysteine completely prevented the cytotoxicity of 16- increased generation of this ROS. We observed that the dehydroprogesterone but not that of the guggulsterone guggulsterone isomers and, to a lesser extent, 16-dehydro- isomers. This observation suggests that either (a) the progesterone indeed generated increased levels of O2 oxidative stress induced by the guggulsterone cannot be before the onset of apoptosis, suggesting that the long-term reversed by 5 mmol/L N-acetylcysteine cotreatment or (b) cytotoxicity of these agents is associated with oxidative the cytotoxicity of 16-dehydroprogesterone depends solely stress. Interestingly, our observations also indicate that at on the generation of ROS. low concentrations (<20 Amol/L) cis-guggulsterone and Finally, we investigated if the guggulsterone and 16- trans-guggulsterone, but not 16-dehydroprogesterone, dehydroprogesterone could induce apoptosis and mito- markedly decreased the pERK expression after treatment chondrial dysfunction in primary CD34-positive leukemia for 48 hours, suggesting that the increased ROS generated cells in culture. Our results show that all three agents by these compounds may contribute to the inactivation of induced rapid (f15 hours) apoptosis in CD34-positive cells ERK signaling (32, 33). Finally, we observed that cytotox- from primary leukemia samples and that this was icity induced by cis-guggulsterone, trans-guggulsterone, associated with mitochondrial dysfunction, although one and 16-dehydroprogesterone was accompanied by in- sample seemed to be more resistant to the cytotoxic effects creased expression of the oxidative stress response protein of these compounds. Most notably, the guggulsterone and heme oxygenase-1, and cis-guggulsterone and trans- 16-dehydroprogesterone were more cytotoxic to leukemia guggulsterone were more effective than 16-dehydropro- blasts than to normal PBMC, suggesting that the pregna- gesterone in inducing this response probably due to their dienedione structure of these agents may display a increased ability to generate ROS. These data indicate therapeutic window. These results are the first to show that oxidative stress is associated with the cytotoxicity the antileukemic activity of the guggulsterone isomers and of the guggulsterone and 16-dehydroprogesterone and 16-dehydroprogesterone in CD34-positive primary leuke- that the guggulsterone can abrogate the activation of ERK mic cells. in leukemic cells. In conclusion, the guggulsterone isomers and 16-dehy- To further investigate the mechanism of action of the droprogesterone represent a novel class of naturally guggulsterone isomers and 16-dehydroprogesterone, we occurring compounds that exhibit antileukemic activity evaluated the short-term effects of higher concentrations by inducing apoptosis and differentiation. Our results (25–75 Amol/L) of these agents. Our results indicate that at indicate that the pregnadienedione structure of these higher concentrations the cytotoxicity of these agents is still steroid isomers has inherent antiproliferative, proapoptotic, associated with apoptosis, mitochondrial dysfunction, and and differentiating activities and may display some ROS generation. In addition, at higher concentrations, 16- selectivity for leukemia blasts over normal PBMC. We are dehydroprogesterone was as effective as the guggulsterone currently investigating the antileukemic effect of synthetic isomers in decreasing pERK levels and this correlated with derivatives of these agents as well as their pharmacokinetic its increased ability to induce ROS at these concentrations. properties in animal models with the goal of developing However, although all three agents induced a dose- and novel and more effective treatments for AML. 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