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Mol Cancer Ther. 2005;4:1982-1992
© 2005 American Association for Cancer Research

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

Ismael Samudio1, Marina Konopleva1, Stephen Safe2,3, Teresa McQueen1 and Michael Andreeff1

1 Section of Molecular Hematology and Therapy, Department of Blood and Marrow Transplantation, The University of Texas M.D. Anderson Cancer Center; 2 Institute of Biosciences and Technology, Texas A&M University, Houston, Texas and 3 Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas

Requests for reprints: Michael Andreeff, Section of Molecular Hematology and Therapy, Department of Blood and Marrow Transplantation, The University of Texas M.D. Anderson Cancer Center, Unit 448, 1400 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-7260; Fax 713-794-474. E-mail: mandreef{at}mdanderson.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, the antileukemic effects of three isomeric pregnadienedione steroids [i.e., cis-guggulsterone, trans-guggulsterone, and 16-dehydroprogesterone] were investigated in HL60 and U937 cells as well as in primary leukemic blasts in culture. Our results show that all three compounds inhibited the proliferation of HL60 and U937 cells, with IC50s ranging from 3.6 to 10.9 µmol/L after treatment for 6 days. These growth inhibitory effects correlated with externalization of phosphatidylserine and loss of mitochondrial membrane potential, suggesting that these isomeric steroids induce apoptosis in leukemia cells. z-VAD-fmk prevented phosphatidylserine externalization but not mitochondrial membrane potential loss, indicating that mitochondrial dysfunction occurred in the absence of caspase activation. Interestingly, although all three compounds increased the generation of reactive oxygen species and decreased phosphorylation of extracellular signal-regulated kinase, only cis-guggulsterone induced a rapid depletion of reduced glutathione levels and oxidation of the mitochondrial phospholipid cardiolipin. 16-Dehydroprogesterone and trans-guggulsterone induced differentiation of HL60 and NB4 cells as evidenced by increased surface expression of CD11b and/or CD14, and all three steroids rapidly induced mitochondrial dysfunction and phosphatidylserine externalization of CD34-positive blasts from primary leukemic samples. This study is the first to show that guggulsterones and 16-dehydroprogesterone exert antileukemic effects via the induction of apoptosis and differentiation and, more importantly, identifies the pregnadienedione structure as a potential chemotherapeutic scaffold. [Mol Cancer Ther 2005;4(12):1982–92]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute myeloid leukemias (AML) are clonal malignancies characterized by increased numbers of immature myeloid progenitor cells arrested at different stages of granulocytic and monocytic differentiation. First-line treatment of AML consists of a combination of cytarabine and an anthracycline, and although this combination results in 60% to 80% complete remissions in newly diagnosed patients, most patients will relapse with resistant disease (1). Because achievement of complete remission is a prerequisite for long-term survival (2), several novel therapeutic modalities have been investigated, including the use of different anthracycline formulations, different nucleoside analogues, and the combination of the antiangiogenic agent thalidomide with cytarabine/anthracycline or topotecan/anthracycline (3–5). However, overall improvement in survival rates has been marginal at best underlining the need for development of more effective therapies. The most striking increase of complete remission and survival has been achieved by ligation of the nuclear retinoic acid receptor {alpha} in acute promyelocytic leukemias with all-trans retinoic acid (6, 7).

The gum resin from the guggul tree Commiphora mukul has been used in Ayurvedic medicine for centuries to treat inflammatory and lipid disorders (8), and an ethylacetate extract of the resin, termed guggulipid, has been reported 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 been shown to lower cholesterol and triglycerides in normal and high-fat-fed rats (9). The antilipidemic effects of guggulsterone may be mediated by antagonism of the orphan receptor FXR (13) as well as promiscuous interactions with other nuclear receptors (14). Notably, although most studies on guggulsterone have focused on their antilipidemic activity, these compounds have also shown potent anti-inflammatory effects, such as preventing oxidative damage during isoproterenol-induced myocardial necrosis in rats (15, 16) and decreasing inflammation associated with nodulocystic acne (17). These observations suggest that in addition to its lipid-lowering activity guggulsterone may modulate anti-inflammatory and antioxidant responses.

A variety of naturally occurring compounds exhibit chemopreventive and anti-inflammatory effects, including resveratrol, betulinic acid, saikosaponin, and curcumin. Some of the chemotherapeutic activities of these compounds may be related to their inhibition of nuclear factor-{kappa}B signaling (18–22), and a recent study reported that cis-guggulsterone inhibited tumor necrosis factor-{alpha}–induced nuclear factor-{kappa}B signaling and sensitized cancer cells to apoptosis induced by taxol, doxorubicin, and tumor necrosis factor-{alpha} (23). Surprisingly, there are no studies to date investigating the direct antiproliferative and proapoptotic effects of guggulsterone in cancer cell lines in culture. We therefore hypothesized that guggulsterone, like other anti-inflammatory and chemopreventive agents, may decrease the proliferation of cancer cells in culture. Here, we report that both isomers of guggulsterone, cis-guggulsterone and trans-guggulsterone, effectively inhibit the proliferation of leukemic cancer cell lines and induce apoptosis and differentiation. Interestingly, a mammalian steroid metabolite and chemical isomer of guggulsterone, 16-dehydroprogesterone, also induced a comparable pattern of differentiation, growth inhibition, and apoptosis, suggesting that the pregnadienedione structure of these steroids (Fig. 1A ) offers the potential for development of novel chemotherapeutics. Our results are the first to show the antileukemic effects of guggulsterone isomers and 16-dehydroprogesterone, and current studies are investigating their mechanism of action and development of more potent novel steroidal analogues.



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  		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 µmol/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.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines, Chemicals, and Biochemicals
U937 and HL60 cells were maintained in RPMI supplemented with 10% FCS, 1% glutamine, and 100 units/mL penicillin in a 37°C incubator containing 5% CO2. 16-Dehydroprogesterone, cis-guggulsterone, and trans-guggulsterone were purchased from Steraloids, Inc. (Newport, RI). TMRM, dihydroethidine, and Cell Tracker Green were all obtained from Molecular Probes (Eugene, OR). z-VAD-fmk was purchased from Alexis Biochemicals (Axxora LLC, San Diego, CA). Phospho–extracellular signal-regulated kinase (ERK) and total ERK antibodies were purchased from Cell Signaling Technologies, Inc. (Beverly, MA). Heme oxygenase-1 antibody was purchased from BD Biosciences (San Jose, CA) and {alpha}-tubulin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals used were of the highest purity available.

Human Subjects
Bone marrow or peripheral blood samples were obtained for in vitro studies from patients with AML. Samples were collected during routine diagnostic procedures after informed consent was obtained in accordance with regulations and protocols approved by the Institutional Review Board of The University of Texas M. D. Anderson Cancer Center (Houston, TX). Mononuclear cells were separated by Ficoll-Hypaque (Sigma Chemical, St. Louis, MO) density gradient centrifugation. Patient sample 1 was a bone marrow aspirate containing 85% blasts from an AML-M1 relapse patient (–7del). Patient sample 2 was a bone marrow aspirate containing 95% blasts from an AML-M1 relapse patient [t(12,17)]. Patient sample 3 was a bone marrow aspirate containing 97% blasts from an AML-M2 relapse patient (normal cytogenetics). Patient sample 4 was a peripheral blood sample containing 92% blasts from an AML-M0 relapse patient [–7del; t(11,19)]. To investigate the effects of the guggulsterone and 16-dehydroprogesterone on normal cells, blood samples from three healthy volunteers (A-C) were obtained and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque density gradient centrifugation. PBMC samples were then exposed to 100 µmol/L guggulsterone and 16-dehydroprogesterone for 20 hours, and phosphatidylserine externalization was quantitated by flow cytometry.

Measurement of Intracellular Glutathione by Flow Cytometry
Cells (3 x 105/mL; 0.5 mL) were treated with compounds as indicated or with 2 mmol/L diethylmaleate for 30 minutes. Cells were then collected by centrifugation, washed in PBS once, and resuspended in 0.2 mL PBS containing 400 µmol/L Cell Tracker Green and incubated at 20°C protected from light for 10 minutes. Cells were then washed in PBS several times, and Cell Tracker Green fluorescence was quantitated by flow cytometry. The mean Cell Tracker Green fluorescence from diethylmaleate-treated samples was considered to be background and subtracted accordingly. All experiments were done in duplicate and repeated at least thrice.

Measurement of Phosphatidylserine Externalization and Mitochondrial Membrane Potential
After appropriate treatments, cells were washed twice in PBS and then resuspended in 100 µL Annexin binding buffer [140 mmol/L NaCl, 10 mmol/L KH2PO4, 5 mmol/L CaCl2 (pH 7.4)] containing 25 nmol/L TMRM and 1:100 dilution of Annexin V-FLUOS (Roche Diagnostics, Mannheim, Germany) incubated at 37°C for 30 minutes. Cells were then analyzed by flow cytometry in a FACSCalibur flow cytometer using a 488 nm argon excitation laser.

Measurement of Reactive Oxygen Species Generation
After appropriate treatments, cells were harvested by centrifugation, washed in PBS, and loaded with the O2–-sensitive probe dihydroethidine. Cells were incubated at 37°C for 10 minutes and washed in PBS, and FL2 fluorescence was examined by flow cytometry. Results presented are means ± SE of three independent experiments.

Western Blot Analysis
Cells where harvested by centrifugation, washed twice in PBS, and resuspended in ice cold lysis buffer [1% Triton X-100, 45 mmol/L KCl, 10 mmol/L Tris (pH 7.5)] supplemented with protease and phosphatase inhibitors and then subjected to SDS-PAGE in 10% or 12% polyacrylamide gels followed by protein transfer to a Hybond-P membrane (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) and immunoblotting. Signals were detected by a PhosphorImager (Storm 860, version 4.0, Molecular Dynamics, Sunnyvale, CA).

Measurement of Cardiolipin Content
After appropriate treatments, cells were harvested by centrifugation, washed once in PBS, and resuspended in PBS containing 10 nmol/L nonyl acridine orange, a probe that binds with high affinity to reduced but not oxidized cardiolipin (24). Cells were incubated 37°C for 30 minutes and FL1 fluorescence was quantitated by flow cytometry.

HL60 and NB4 Cell Differentiation
HL60 and NB4 cells were treated with 10 µmol/L guggulsterone and 16-dehydroprogesterone, and after 120 hours, the cells were collected, washed once in PBS, and resuspended in PBS containing 1:100 dilution of CD11b-phycoerythrin and CD14-FITC (both form BD Biosciences). Cells were incubated at room temperature for 15 minutes, washed in PBS, and analyzed by flow cytometry gating on viable cells as determined by forward and side scatter. Cells stained with mouse IgG-phycoerythrin and mouse IgG-FITC served as negative controls. In parallel, viable cells were counted in a hemocytometer after trypan blue exclusion. The absolute number of differentiated cells was calculated from the equation:

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Guggulsterone Isomers and 16-Dehydroprogesterone Prevent the Growth of Leukemic Cells in Culture by Inducing Apoptosis
Because there are no reports in the literature investigating the antiproliferative effects of guggulsterone or related pregnadienediones, we cultured HL60 and U937 cells with increasing concentrations of both guggulsterone isomers and 16-dehydroprogesterone for 72 and 144 hours and quantitated the number of viable cells remaining after treatment. The results in Fig. 1B show that cis-guggulsterone, trans-guggulsterone, and 16-dehydroprogesterone inhibited the proliferation of HL60 cells in a time- and dose-dependent manner displaying 144-hour IC50s ranging from 8.3 to 10.9 µmol/L. Similarly, all three compounds inhibited the proliferation of U937 cells with slightly higher potencies displaying IC50s varying from 3.6 to 8.7 µmol/L (Fig. 1C). To investigate whether apoptosis contributed to the antiproliferative effects of the guggulsterone and 16-dehydroprogesterone, we quantitated the percentage of HL60 and U937 cells that externalized phosphatidylserine after treatment with these agents for 72 hours. We found that all three compounds significantly increased phosphatidylserine externalization, albeit cis-guggulsterone was the most potent compound displaying 72-hour IC50s of 16.1 and 19.8 µmol/L after treatment of HL60 and U937 cells, respectively, for 72 hours (Fig. 2A ). Furthermore, the increase in phosphatidylserine externalization correlated with a marked loss in mitochondrial membrane potential ({Delta}{Psi}m) as evidenced by reduced accumulation of the potentiometric probe TMRM, which was more pronounced in cells treated with cis-guggulsterone (Fig. 2B). Taken together, these results show that the isomeric pregnadienedione steroids guggulsterone and 16-dehydroprogesterone prevent the proliferation of leukemic cells in culture partly by inducing mitochondrial dysfunction and apoptosis.



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  		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 µmol/L) for 72 h. Phosphatidylserine externalization and {Delta}{Psi}m 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 µmol/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 µmol/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.

 
Guggulsterone Isomers and 16-Dehydroprogesterone Induce Differentiation of HL60 and NB4 Cells in Culture
HL60 cells have been shown to differentiate in culture after treatment with several anticancer drugs and apoptosis inducers with lipophilic and/or steroidal structure, and these include the synthetic triterpenoid CDDO-Me, 12-O-tetradecanoylphorbol-13-acetate, oxysterols, and 1,25-dihydroxyvitamin D3 (25–30). Because one mechanism that may contribute to the antiproliferative effects of the guggulsterone and 16-dehydroprogesterone is the induction of differentiation, we examined the surface expression of the monocytic and myelomonocytic markers CD14 and CD11b, respectively, in HL60 cells treated with 10 µmol/L 16-dehydroprogesterone, cis-guggulsterone, and trans-guggulsterone for 120 hours. Of note, because the selective killing of immature cells by these steroids would increase the relative numbers of differentiated cells, we calculated the absolute number of cells expressing surface CD14 or CD11b as described in Materials and Methods. These results presented in Fig. 2C illustrate that trans-guggulsterone was the more potent inducer of myelomonocytic differentiation, promoting a 2.5-fold increase in HL60 cells expressing surface CD11b (P < 0.004) followed by 16-dehydroprogesterone, which induced a modest but not statistically 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-guggulsterone promoted an increase in cells expressing this monocytic marker. Interestingly, cis-guggulsterone seemed to decrease the numbers of cells expressing both surface markers probably owing to its higher cytotoxicity at 10 µmol/L compared with 16-dehydroprogesterone or trans-guggulsterone. Finally, we also investigated if these steroids would promote differentiation in a different cell context. For these experiments, we treated the acute promyelocytic leukemia cell line NB4, which has been shown to undergo monocytic differentiation after treatment with retinoic acid and 1,25-dihydroxyvitamin D3 (28–30), with 10 µmol/L guggulsterone and 16-dehydroprogesterone for 96 hours and examined the cell surface expression of CD14 (Fig. 2D). 16-Dehydroprogesterone and trans-guggulsterone induced a robust increase in NB4 cells expressing surface CD14 (3.8- and 3.5-fold, respectively; P < 0.008), whereas cis-guggulsterone induced a more modest but significant 1.7-fold increase (P < 0.03). Under these conditions, 16-dehydroprogesterone and trans-guggulsterone decrease the number of viable NB4 cells by ~50%, whereas cis-guggulsterone induced a more pronounced ~80% decrease (data not shown). Notably, we have observed that lower concentrations of cis-guggulsterone (<10 µmol/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 µmol/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% increase in O2– (Fig. 3A ). Under these conditions, there was minimal apoptosis (data not shown), suggesting that the observed increase in O2– was not a consequence of cell death. Previous reports have shown that induction of apoptosis in U937 cells by agents, such as CDDO-Me, bortezomib, adaphostin, and arsenic trioxide, which induce oxidative stress, is accompanied by inhibition of ERK phosphorylation (31–34). We therefore investigated if the effect of guggulsterone isomers and 16-dehydroprogesterone on the levels of phosphorylated ERK (pERK) in U937 cells by Western blot. Guggulsterone isomers induced a ~90% decrease in the levels of pERK after 48 hours compared with cells treated with vehicle (DMSO). In contrast, U937 cells treated with 16-dehydroprogesterone only exhibited a ~ 20% inhibition of ERK phosphorylation. In addition, the enhanced formation of O2– promoted by the guggulsterone isomers and 16-dehydroprogesterone was associated with increased expression of the oxidative stress responsive gene heme oxygenase-1 (Fig. 3B), and this increase was greater in cells treated with cis-guggulsterone (38-fold) and trans-guggulsterone (32-fold) than in cells treated with 16-dehydroprogesterone (3-fold). Thus, guggulsterone isomers and, to a lesser extent, 16-dehydroprogesterone induce oxidative stress in U937 cells that is accompanied by decreased activation of ERK and increased levels of heme oxygenase-1.



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  		Figure 3. Guggulsterone isomers and 16-dehydroprogesterone induced the generation of O2–, decrease the activation of ERK, and induce expression of the stress response protein heme oxygenase-1. A, U937 cells were treated with 20 µmol/L cis-guggulsterone, trans-guggulsterone, and 16-dehydroprogesterone for 24 h and the levels of O2– were quantitated by flow cytometry as described in Materials and Methods. B, U937 cells were treated with 15 µmol/L cis-guggulsterone, trans-guggulsterone, or 16-dehydroprogesterone for 48 h and the levels of pERK, total ERK, heme oxygenase-1 (HO-1), and {alpha}-tubulin were examined by Western blot as described in Materials and Methods. Superoxide measurements were done in triplicate and repeated at least twice. Columns, mean of a representative experiment; bars, SD. *, P < 0.05; **, P < 0.0005.

 
Rapid Cytotoxicity of Higher Concentrations of Guggulsterone Isomers and 16-Dehydroprogesterone Is Associated with ROS Generation, Inactivation of ERK, and Induction of Apoptosis
To further investigate the short-term cytotoxic effects of the guggulsterone isomers and 16-dehydroprogesterone, we treated U937 cells with higher concentrations of these agents (25–75 µmol/L) for 20 hours. Our results show that, as observed for lower concentrations (10–20 µmol/L) of guggulsterone isomers and 16-dehydroprogesterone, higher concentrations of these agents also induced a dose-dependent externalization of phosphatidylserine, loss of {Delta}{Psi}m, and increased generation of O2– (Fig. 4A ), suggesting a priori that at higher concentrations these compounds elicit similar cytotoxic responses albeit displaying faster kinetics. Moreover, 16-dehydroprogesterone was as effective as guggulsterone isomers in decreasing the levels of pERK after treatment with 50 µmol/L for 20 hours and this correlates with its comparable ability to induce ROS at these concentrations. The 50 µmol/L concentrations of all compounds induced heme oxygenase-1 expression and cleavage of caspase-3, suggesting that the observed oxidative stress correlates with caspase activation. To further investigate the kinetics of oxidative stress induced by 16-dehydroprogesterone and the guggulsterone, we quantitated the generation of O2– 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) accumulation of O2– (6.6- and 5.3-fold, respectively), whereas cis-guggulsterone failed to significantly increase the levels of O2– (P > 0.05; Fig. 4B). Increased generation of ROS by 16-dehydroprogesterone and trans-guggulsterone was sustained for 6 hours, and at this time, cis-guggulsterone showed a significant (P < 0.003) 7.8-fold increase in the O2– levels, suggesting that, although all three agents provoke ROS generation, they induce this response with different kinetics.



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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 µmol/L) for 20 h, and phosphatidylserine externalization, {Delta}{Psi}m, and O2– generation were quantitated as described in Materials and Methods. In addition, the levels of pERK, total ERK, heme oxygenase-1, and {alpha}-tubulin were examined. B, U937 cells were treated with 75 µmol/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 µmol/L cis-guggulsterone, trans-guggulsterone, and 16-dehydroprogesterone for 3 and 6 h and intracellular glutathione was quantitated as described in Materials and Methods. D, U937 cells were treated with increasing concentrations of the guggulsterone and 16-dehydroprogesterone (25–75 µmol/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.

 
Cytotoxic Concentrations of the Guggulsterone Isomers and 16-Dehydroprogesterone Uncover a Selective Depletion of Reduced Glutathione and Oxidation of Cardiolipin Induced by cis-Guggulsterone
Because reduced glutathione levels are critical determinants of intracellular redox homeostasis (35), we also investigated the effects of guggulsterone isomers and 16-dehydroprogesterone on the levels of this intracellular antioxidant in U937 cells. Interestingly, at 3 hours, cis-guggulsterone induced a significant (P < 0.0002) 24% decrease in the levels of intracellular glutathione, whereas trans-guggulsterone, which induced significant increases in ROS at this time point, failed to affect the levels of glutathione; U937 cells treated with 16-dehydroprogesterone for 3 hours only displayed a slight albeit significant (P < 0.003) 7% decrease in glutathione (Fig. 4C). The decrease in glutathione induced by cis-guggulsterone was maintained 6 hours after treatment, and at this time, neither 16-dehydroprogesterone nor trans-guggulsterone elicited a significant decrease (P > 0.05) in the levels of this antioxidant in U937 cells. Because cis-guggulsterone decreased the levels of intracellular glutathione, we investigated if this pregnadienedione would promote oxidation of the mitochondrial phospholipid cardiolipin. Cardiolipin is essential for mitochondrial function and for preventing apoptosis by sequestering cytochrome c (36, 37). Glutathione is required for maintaining appropriate levels of reduced cardiolipin via the action of a glutathione-dependent peroxidase that is antiapoptotic (38). Indeed, consistent with the effects of cis-guggulsterone on glutathione, the results presented in Fig. 4D show that after 20 hours treatment cis-guggulsterone induced a dramatic increase in the percentage of U937 cells with low levels of cardiolipin, whereas 16-dehydroprogesterone and trans-guggulsterone failed to elicit a similar response. Taken together, these results show that, although all three pregnadienediones induce rapid generation of ROS in U937 cells, they display different kinetics and only cis-guggulsterone provokes marked decreases in glutathione and substantial loss of cardiolipin.

Mitochondrial Dysfunction Induced by the Guggulsterone Isomers and 16-Dehydroprogesterone Is Independent of Caspase Activation
Because it has been reported recently that caspases mediate loss of {Delta}{Psi}m induced by a variety of proapoptotic stimuli (39, 40), we investigated if mitochondrial dysfunction and apoptosis induced by 50 µmol/L guggulsterone and 16-dehydroprogesterone after 24 hours was dependent on the activity of these proteases. In addition, we also investigated if the potent antioxidant N-acetylcysteine could prevent cytotoxicity induced by guggulsterone and 16-dehydroprogesterone. The results presented in Fig. 5A show that loss of {Delta}{Psi}m induced by all three compounds was not affected by pharmacologic inhibition of caspases using the pancaspase inhibitor z-VAD-fmk. In contrast, z-VAD-fmk significantly (P < 0.001) prevented the externalization of phosphatidylserine induced by all three compounds, suggesting that caspase inhibition in cells treated with guggulsterone and 16-dehydroprogesterone switches the mode of cell death from apoptosis to necrosis (Fig. 5B). Interestingly, the antioxidant N-acetylcysteine completely prevented the cytotoxicity induced by 16-dehydroprogesterone but not that induced by cis-guggulsterone or trans-guggulsterone, suggesting that 16-dehydroprogesterone may depend solely on ROS to induce cell death. These data illustrate that the cis-guggulsterone and trans-guggulsterone, but not 16-dehydroprogesterone, induce cytotoxicity independent of the generation of ROS and that all three compounds induce mitochondrial dysfunction in the absence of caspase activation, but caspases contribute to the onset of apoptosis in cells treated with these agents.



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  		Figure 5. Mitochondrial dysfunction induced by the guggulsterone isomers and 16-dehydroprogesterone is independent of caspase activation and the antioxidant N-acetylcysteine (NAC) only prevents cell death induced by 16-dehydroprogesterone but not by cis-guggulsterone or trans-guggulsterone. Briefly, U937 cells were treated with 50 µmol/L cis-guggulsterone, trans-guggulsterone, or 16-dehydroprogesterone alone or in combination with the pancaspase inhibitor z-VAD-fmk (50 µmol/L) or the potent antioxidant N-acetylcysteine (5 mmol/L), and {Delta}{Psi}m (A) and phosphatidylserine externalization (B) were quantitated after 24 h.

 
Guggulsterone Isomers and 16-Dehydroprogesterone Induce Mitochondrial Dysfunction and Apoptosis in CD34-Positive Cells from Primary Leukemia Samples
To determine if the guggulsterone and 16-dehydroprogesterone would induce apoptosis in CD34-positive cells from primary leukemia samples, we exposed ex vivo four primary leukemic samples to increasing concentrations (25–100 µmol/L) of these agents for 15 hours and examined externalization of phosphatidylserine in CD34-positive cells by flow cytometry. For three samples, we also investigated the loss of {Delta}{Psi}m in CD34-positive cells. Our results illustrated in Fig. 6A show that 25 µmol/L guggulsterone isomers and 16-dehydroprogesterone induced phosphatidylserine externalization in CD34-positive blasts from all samples tested (P < 0.02), albeit sample 3 was markedly more resistant to the cytotoxicity of all three compounds. Furthermore, all three compounds induced significant (P < 0.03) decreases in {Delta}{Psi}m in CD34-positive blasts from patients 2 to 4 (Fig. 6B). Finally, we investigated the cytotoxicity of 100 µmol/L guggulsterone and 16-dehydroprogesterone in normal PBMCs obtained from healthy volunteers. As shown in Fig. 6C, none of the three agents induced apoptosis in normal PBMC to the same extent as in leukemia blasts. Notably, trans-guggulsterone was the least cytotoxic pregnadienedione to PBMC minimally increasing phosphatidylserine externalization above DMSO-treated cells by an average of 2.1 ± 5.4% compared with 50.7 ± 21.6% in leukemia blasts (P < 0.02). Similarly, 16-dehydroprogesterone induced significantly less apoptosis in normal PBMC than in leukemia blasts (11.5 ± 6% versus 47.9 ± 20.7%; P < 0.04). cis-Guggulsterone exhibited the highest cytotoxicity in PBMC increasing phosphatidylserine externalization by 22.5 ± 7.7%, albeit this increase was significantly lower (P < 0.05) than the observed 60.7 ± 23.5% increase in leukemia blasts treated with this agent. Taken together, these findings show that guggulsterone isomers and 16-dehydroprogesterone effectively induce apoptosis in CD34-positive cells from primary leukemia samples but not in normal PBMC and that apoptosis induced in leukemia blasts is associated with marked mitochondrial dysfunction.



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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 µmol/L) of cis-guggulsterone, trans-guggulsterone, or 16-dehydroprogesterone for 15 h. Phosphatidylserine externalization (A) and {Delta}{Psi}m (B) were quantitated in CD34-positive cells. C, PBMC samples from three healthy volunteers (A–C) were exposed to 100 µmol/L guggulsterone and 16-dehydroprogesterone for 20 h and phosphatidylserine externalization was quantitated by flow cytometry.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Natural products have provided a large number of currently 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 anticancer properties (43), and many active biological components have been chemically modified to generate promising new chemotherapeutic drugs (44–46). We have shown previously that synthetic derivatives of oleanolic acid and diindolylmethane, found in the oleander tree and cruciferous vegetables, respectively, potently induced apoptosis in leukemic cell lines and primary leukemic samples (26, 47, 48). The naturally occurring plant sterols, the guggulsterones, are the active components of the antilipidemic extract of the guggul tree C. mukul that are currently being evaluated for treatment of hypercholesterolemia and obesity (8, 10, 11). Interestingly, these pregnane sterols also possess anti-inflammatory activity that may be in part dependent on their ability to inhibit nuclear factor-{kappa}B signaling (15–17, 23). We thus hypothesized a priori that like other nuclear factor-{kappa}B 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 µmol/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 compounds significantly increased the number of NB4 cells expressing CD14, suggesting that these steroids can promote monocytic differentiation in a cell context–dependent manner. These are the first data that show the antiproliferative, proapoptotic, and differentiating effects of the guggulsterone isomers and 16-dehydroprogesterone in leukemic cells in culture.

Because the generation of O2– is an early event in many forms of cell death and is an indicator of mitochondrial dysfunction (49, 50), we also examined if the cytotoxicity of low concentrations (<20 µmol/L) of the guggulsterone isomers and 16-dehydroprogesterone was associated with increased generation of this ROS. We observed that the guggulsterone isomers and, to a lesser extent, 16-dehydroprogesterone indeed generated increased levels of O2– before the onset of apoptosis, suggesting that the long-term cytotoxicity of these agents is associated with oxidative stress. Interestingly, our observations also indicate that at low concentrations (<20 µmol/L) cis-guggulsterone and trans-guggulsterone, but not 16-dehydroprogesterone, markedly decreased the pERK expression after treatment for 48 hours, suggesting that the increased ROS generated by these compounds may contribute to the inactivation of ERK signaling (32, 33). Finally, we observed that cytotoxicity induced by cis-guggulsterone, trans-guggulsterone, and 16-dehydroprogesterone was accompanied by increased expression of the oxidative stress response protein heme oxygenase-1, and cis-guggulsterone and trans-guggulsterone were more effective than 16-dehydroprogesterone in inducing this response probably due to their increased ability to generate ROS. These data indicate that oxidative stress is associated with the cytotoxicity of the guggulsterone and 16-dehydroprogesterone and that the guggulsterone can abrogate the activation of ERK in leukemic cells.

To further investigate the mechanism of action of the guggulsterone isomers and 16-dehydroprogesterone, we evaluated the short-term effects of higher concentrations (25–75 µmol/L) of these agents. Our results indicate that at higher concentrations the cytotoxicity of these agents is still associated with apoptosis, mitochondrial dysfunction, and ROS generation. In addition, at higher concentrations, 16-dehydroprogesterone was as effective as the guggulsterone isomers in decreasing pERK levels and this correlated with its increased ability to induce ROS at these concentrations. However, although all three agents induced a dose- and time-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 a different mechanism to induce oxidative stress. Notably, only cis-guggulsterone markedly decreased the levels of cardiolipin, suggesting that the decrease in glutathione induced by this agent may lead to oxidation of this critical mitochondrial phospholipid. The use of higher concentrations of the guggulsterone and 16-dehydroprogesterone uncovered a cis-guggulsterone-specific effect on the levels of glutathione and cardiolipin in U937 cells, suggesting that the cytotoxicity of cis-guggulsterone may be mediated by a different mechanism from that of trans-guggulsterone or 16-dehydroprogesterone.

We also investigated if caspases were involved in the cytotoxicity of the guggulsterone and 16-dehydroprogesterone and found that pharmacologic inhibition of these proteases with z-VAD-fmk prevented phosphatidylserine externalization but not loss of {Delta}{Psi}m, suggesting that mitochondrial dysfunction induced by these agents occurs before caspase activation but caspases contribute to the induction of apoptosis. The potent antioxidant N-acetylcysteine completely prevented the cytotoxicity of 16-dehydroprogesterone but not that of the guggulsterone isomers. This observation suggests that either (a) the oxidative stress induced by the guggulsterone cannot be reversed by 5 mmol/L N-acetylcysteine cotreatment or (b) the cytotoxicity of 16-dehydroprogesterone depends solely on the generation of ROS.

Finally, we investigated if the guggulsterone and 16-dehydroprogesterone could induce apoptosis and mitochondrial dysfunction in primary CD34-positive leukemia cells in culture. Our results show that all three agents induced rapid (~15 hours) apoptosis in CD34-positive cells from primary leukemia samples and that this was associated with mitochondrial dysfunction, although one sample seemed to be more resistant to the cytotoxic effects of these compounds. Most notably, the guggulsterone and 16-dehydroprogesterone were more cytotoxic to leukemia blasts than to normal PBMC, suggesting that the pregnadienedione structure of these agents may display a therapeutic window. These results are the first to show the antileukemic activity of the guggulsterone isomers and 16-dehydroprogesterone in CD34-positive primary leukemic cells.

In conclusion, the guggulsterone isomers and 16-dehydroprogesterone represent a novel class of naturally occurring compounds that exhibit antileukemic activity by inducing apoptosis and differentiation. Our results indicate that the pregnadienedione structure of these steroid isomers has inherent antiproliferative, proapoptotic, and differentiating activities and may display some selectivity for leukemia blasts over normal PBMC. We are currently investigating the antileukemic effect of synthetic derivatives of these agents as well as their pharmacokinetic properties in animal models with the goal of developing novel and more effective treatments for AML. The understanding of the mechanism of action of this novel class of steroidal compounds will offer additional targets for the treatment of human leukemias.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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