Dehydroleucodine, a Sesquiterpene Lactone from ... - ACS Publications

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Dehydroleucodine, a Sesquiterpene Lactone from Gynoxys verrucosa, Demonstrates Cytotoxic Activity against Human Leukemia Cells Paola E. Ordóñez,†,§,∥ Krishan K. Sharma,‡ Laura M. Bystrom,‡ Maria A. Alas,‡ Raul G. Enriquez,⊥ Omar Malagón,§ Darin E. Jones,∥ Monica L. Guzman,*,‡ and Cesar M. Compadre*,† †

Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, United States Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medical College, New York, New York 10065, United States § Departamento de Química, Universidad Técnica Particular de Loja, Loja, Ecuador ⊥ Instituto de Química, Universidad Nacional Autónoma de México, México DF, México ∥ Department of Chemistry, University of Arkansas at Little Rock, Little Rock, Arkansas 72205, United States ‡

S Supporting Information *

ABSTRACT: The sesquiterpene lactones dehydroleucodine (1) and leucodine (2) were isolated from Gynoxys verrucosa, a species used in traditional medicine in southern Ecuador. The activity of these compounds was determined against eight acute myeloid leukemia (AML) cell lines and compared with their activity against normal peripheral blood mononuclear cells. Compound 1 showed cytotoxic activity against the tested cell lines, with LD50 values between 5.0 and 18.9 μM. Compound 2 was inactive against all of the tested cell lines, demonstrating that the exocyclic methylene in the lactone ring is required for cytotoxic activity. Importantly, compound 1 induced less toxicity to normal blood cells than to AML cell lines and was active against human AML cell samples from five patients, with an average LD50 of 9.4 μM. Mechanistic assays suggest that compound 1 has a similar mechanism of action to parthenolide (3). Although these compounds have significant structural differences, their lipophilic surface signatures show striking similarities.

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Naturally occurring compounds are one of the most promising sources of new drugs for cancer treatment. Their large structural diversity and compatibility with biological systems make them one of the best sources of therapeutic agents for cancer treatment. A recent review reports that 49% of the small molecules approved for cancer from the 1940s to 2014 are either natural products or directly derived from natural products.6 Found in many plants, sesquiterpene lactones (SLs) are a class of compounds exhibiting a wide range of biological activities such as anti-inflammatory, antiparasitic, antibacterial, and cytotoxic effects.7−11 Parthenolide (3), a SL extracted from the medicinal herb feverfew (Tanacetum parthenium), possesses antileukemic activity. Compound 3 induces several intracellular effects, including the generation of reactive oxygen species and the inhibition of the nuclear factor kappa-light-chain-enhancer of

cute myeloid leukemia (AML) is a fatal disease with an overall five-year survival rate of 19.8%.1 In the United States, over 12 000 adults are diagnosed with AML each year.2 The treatment regimens for AML patients have remained relatively stagnant for the last three decades.1 Despite achieving complete remission after aggressive multiagent chemotherapy and allogeneic stem cell transplantation, most patients relapse and die of this disease.3 AML is a genetically heterogeneous disease consisting of leukemic stem, progenitor, and blast cell populations. Common chemotherapeutic agents, such as cytosine arabinoside (ara-C), target only actively cycling cells; thus, they fail to eliminate quiescent leukemic stem cell (LSC) populations.4 The failure to eliminate LSCs is thought to provide a reservoir for disease relapse. To effectively target all of the AML subpopulations, including leukemic stem and progenitor cells, new chemotherapeutic drugs are urgently required. Furthermore, common chemotherapy is significantly toxic and can have adverse side effects.5 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 2, 2015

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DOI: 10.1021/acs.jnatprod.5b00383 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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activated B cells (NF-κB), which are critical for the survival of AML stem and progenitor cells.4,12,13 However, the potential clinical applications of 3 have been hindered because of its limited stability,14 water solubility,15,16 and bioavailability.12 Recently it has been shown that other SLs can inhibit AML stem and progenitor cell growth.8 However, the structural characteristics of SLs required to target all of the AML subpopulations, including stem cells, remain unclear. Thus, it would be advantageous to discover and evaluate the antileukemic potential of additional SLs for therapeutic purposes and for mechanistic studies.

In this context, the activity of the extracts from Gynoxys verrucosa V.M. Badillo, a shrub from the family Asteraceae, was tested against a battery of AML cells. The aerial parts of this plant, commonly known as guángalo, are used in traditional medicine in southern Ecuador to treat skin infections and to promote wound healing by direct application to the skin.9,17 From the extract exhibiting activity against leukemia cells two SLs were isolated: dehydroleucodine (1), which shows cytotoxic activity, and leucodine (2), which has no discernible activity. In this study it was also determined that 1 inhibited the activation of NF-κB in a manner similar to 3.

Figure 1. ORTEP projections of the X-ray crystal structure of compound 2 with 50% probability ellipsoids.

Table 1. Cytotoxic Effects of Compounds 1−3 and the G. verrucosa Extract, Expressed as LD50 Valuesa

HL-60 Kasumi-1 KG-1 MOLM-13 MV4-11 THP-1 TUR U937

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RESULTS AND DISCUSSION Compounds 1 and 2 were isolated from G. verrucosa by following a previously reported procedure.9 The X-ray crystal structure of 2 was determined, demonstrating that its structure is similar to that of compound 1, except for the presence of two hydrogens at C-13 and C-12. The hydrogen attached to C-12 is above the approximate plane of the seven-membered ring. Therefore, the relative configuration of 2 is confirmed and it is consistent with the previously reported single X-ray diffraction analysis of this compound.18 However, in this research the absolute configuration for molecule 2 was determined based on its Flack parameter (0.01(4)) as 8S, 9S, 10S, and 12S; a perspective ORTEP plot is shown in Figure 1. The results in Table 1 show the activity of the G. verrucosa extract as well as compounds 1−3 against eight human leukemia cell lines. Although this is the first report of the potential antileukemic activity of 1, its activity has been evaluated in other models including KB (IC50 of 5.3 μM),19 HeLa S3 (IC50 of 10.0 μM), and MCF-7 (IC50 of 5.0 μM) cell lines.20 Compound 1 has also shown an effect on the proliferation of B16 melanoma and Melan-A cells,21 and it has inhibited the activation of LAD2 mast cells.22 As shown in Table 1, compound 1 has cytotoxic activity against multiple AML cell lines after 48 h of treatment, with LD50 values ranging from 5.0 to 18.9 μM. Compound 2 was inactive against all of the leukemia cell lines, demonstrating that the exocyclic methylene in 1 is required for the observed activity. Table 1 shows that the activity profiles of 1 and 3 are similar. However, 3 was inactive against the KG-1 and THP-1 cell lines, while 1 was active against all the cell lines. These results show clearly that structural modification of the SLs

a

G. verrucosa EtOAc extract (μg/ mL)

1 (μM)

2 (μM)

3 (μM)

4.3 3.5 7.1 3.1 3.0 7.0 5.7 6.0

14.1 12.9 18.7 12.6 5.0 16.8 12.2 18.9

>20 >20 >20 >20 >20 >20 >20 >20

13.9 3.6 >20 3.4 8.6 >20 7.4 8.4

Tested range for pure compounds: 1.25−160 μM.

could dramatically increase their specificity against particular cell populations. When determining the potential use of a new chemotherapeutic agent in patients, it is desirable for the drug to effectively target tumor cells without causing significant harm to noncancerous cells. Thus, to better establish the potential of 1 as an antileukemic drug, its activity and that of compound 2 were tested on normal bone marrow (n = 1) and peripheral blood (n = 3) mononuclear cells. Figure 2A displays the viability of the normal samples after 48 h of treatment with 20 μM dehydroleucodine (1). Compound 3 and the G. verrucosa extract were included as references, compound 3 being a wellknown anticancer compound.12,13 In a similar way to when tested against AML cell lines 2 displayed no toxicity against normal cells. Importantly, Figure 2B shows that 1 was significantly more potent to the tested AML cell lines (n = 8) than to normal mononuclear cells (n = 4, p = 0.004). These data strongly suggest that 1 represents a promising chemotherapeutic agent. To better understand the mechanism by which 1 kills AML cells, the intracellular effects upon treatment with compounds 1 and 2 were tested using quantitative PCR (Figure 3). Leukemia cells were treated with 20 μM of each test compound for 6 h before RNA extraction. As the reported mechanism of action of 3 involves an increase in oxidative stress and inhibition of NFB



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Figure 2. Compound 1 is significantly less potent in normal peripheral blood mononuclear cells (PBMNCs) than in leukemic cells. (A) Viability of normal cells after treatment with 1 and its derivatives. Bone marrow and PBMNCs were isolated from healthy donors and treated for 48 h with 20 μM compound 1 or 3 or with 50 μM compound 2. Cells were also treated with 10 μg/mL G. verrucosa extract as a reference. Viability was evaluated by annexin V and 7AAD staining using flow cytometry. Each line represents a distinct sample with viabilities calculated relative to an untreated control. (B) Average viability of AML cell lines compared with normal BM/PBMNCs treated with 20 μM compound 1 after 48 h. The percent viabilities of the eight cell lines from Table 1 at 20 μM compound 1 were averaged and graphed next to the average percent viability of the four healthy donor samples. The significance between the two groups was calculated using the Mann−Whitney test, p = 0.004.

Figure 3. Compound 1 induced HMOX1 and HSPA1A and downregulated NF-κB. This graphic represents the fold changes of (A) HMOX1, (B) HSPA1A, and (C) NFkB1A gene expression in MOLM-13 cells. Experiments were performed in triplicate. Lines represent the mean for each specimen, with error bars representing the SD. The fold changes were calculated by the delta−delta Ct method. (D) DNA-binding ELISAs for NFκB (p65) activity for compounds 1−3. TNFα was used as a positive control. Experiments were performed in triplicate. Lines represent the mean for each specimen, with error bars representing the SD. The fold changes were calculated by the delta−delta Ct method. (E) Fold change in gene expression for known NF-κB target genes (CDK6, MYB). (F) Immunoblot for MOLM-13 cells after 6 h of treatment with either compound 1 or 3 at 5 or 10 μM. The blot was probed for phospho-p65 and p65 antibodies. Total p65 is shown as the loading control.

κB,12 the ability of 1 to perturb these pathways was studied. Heme oxygenase 1 (HMOX1) and the primary stress-inducible isoform of Hsp70 (HSPA1A) were investigated to determine the activation of an oxidative stress response upon drug treatment (Figure 3A and B, respectively). It was found that 1 upregulated HMOX1 and HSPA1A, as observed with 3,

suggesting that compound 1 can activate oxidative stress responses as reported for compound 3.12 However, the higher fold changes by treatment with 1 suggest that this compound may induce a greater amount of oxidative stress than 3. Furthermore, compound 2 moderately upregulated HMOX1 and HSPA1A. C



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mented in SYBYLX 2.1.1 (Certara, St. Louis, MO, USA) was performed. Figure 5 compares the lipophilic surface signatures of compounds 1 and 3, showing a strong and clearly delineated

Compound 3 has been shown to induce apoptosis on leukemic stem cells and progenitor cells through inhibition of NF-κB.5 Thus, the levels of NFKB1A transcript upon drug treatment (Figure 3C) were evaluated. Treatment with compounds 1−3 resulted in downregulation of NFKB1A transcription. To better understand their effects on the activity of NF-κB, compounds 1 (the lead compound from G. verrucosa), 2 (inactive against leukemia cells and lacking the exocyclic methylene), and 3 (reported NF-κB inhibitor) were studied using a DNA-binding ELISA assay to directly evaluate the DNA-binding activity of NF-κB. From this analysis, it was found that compounds 1 and 3 could inhibit NF-κB (Figure 3D), while compound 2 does not significantly affect its activity. In addition, the levels of phospho-p65 and total p65 upon drug treatment were evaluated using a Western blot analysis (Figure 3D). This activity was confirmed by evaluating other reported NF-κB target genes (MYB and CDK6)23 (Figure 3E). The p65 transcription factor is involved in NF-κB heterodimer formation, and the phosphorylation of p65 is essential for NF-κB activation. Strikingly, 1 and 3 induced a significant decrease in phospho-p65, as shown in Figure 3F, where total p65 is shown as a control. Owing to its mechanistic similarities to compound 3 and its ability to decrease phospho-p65, compound 1 may be useful in the treatment of leukemic progenitor and stem cells. The use of tumor-derived cell lines, such as those used to screen the activity of compound 1, presents many practical advantages. However, these cell lines may not be representative of the cell phenotypes encountered when evaluating potential drugs in vivo.24 Thus, to further establish the potential of compound 1 to treat AML, its cytotoxic activity was tested against primary leukemia cells isolated from seven different AML patients. Figure 4 shows that compound 1 induced cell death in all of the primary samples tested, with an average LD50 of 9.4 μM.

Figure 5. (A) X-ray crystal structures of 1 and 3 and (B) their corresponding lipophilic surface signatures.

lipophilic area at the top of the convex part of the molecules and a more hydrophilic surface at the bottom of the concave part of the structures. Interestingly, the convex lipophilic part of the molecule is where the highest similarity occurs, and the concave part is where there are the greatest differences. The implication of these findings is that the SL receptor should have a complementary lipophilic area, possibly explaining why these substantially different compounds appear to interact with the same receptors. Although, 1 is mechanistically very similar to 3, compound 1 has some advantages that bode well for its potential development as an antileukemic drug. Compound 1 showed cytotoxicity against AML cell lines and against human AML cell samples from patients. As shown in Figure 2A, compound 1 is slightly less toxic to normal cells than 3, but as shown in Table 1, compound 1 is more toxic than 3 to most of the AML cell lines. Importantly, the epoxide ring that is responsible for the instability of 328 is not present in compound 1. Additionally, 1 is present in high yield (0.35%), and it is easily isolated from G. verrucosa as an abundant plant species.

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Figure 4. Activity of dehydroleucodine (1) in primary samples compared with cytosine arabinoside (Ara-C). Each symbol represents a primary AML sample tested (compound 1, n = 7; Ara-C, n = 5).

EXPERIMENTAL SECTION

General Experimental Procedures. Column chromatography was performed using 60−230 mesh silica gel. Preparative TLC was performed on precoated silica gel 60 F254 plates (0.2 mm thick, EMD Millipore, Billierica, MA, USA). Plant Material. Gynoxys verrucosa was collected in June 2007 in the locality of Yangana, in the Loja Province of Ecuador. The plant material was identified by Vladimir Morocho, Ph.D., and voucher specimens (PPN-as-11) are deposited at the Herbarium of the Chemistry Department of the Universidad Técnica Particular de Loja, Loja Ecuador, and at the Herbarium Reynaldo Espinoza of the Universidad Nacional de Loja. Extraction and Isolation. The dried aerial parts of G. verrucosa were extracted with ethyl acetate at room temperature and concentrated under reduced pressure. To remove the chlorophylls, the crude extract was filtered through a reversed-phase C18 column

Compounds 1 and 3 have considerable structural differences. For example, compound 3 has a 10-membered ring attached to a five-membered ring, while compound 1 has a five-membered ring attached to a seven-membered ring, which is attached to another five-membered ring. However, the fact that these compounds appear to have the same mechanism of action suggests that they possess chemical similarities that are recognizable by their receptors. To identify those similarities, a surface signature analysis of both molecules using their X-ray crystal structures9,25 and the MOLCAD program as impleD



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with a mixture MeOH−H2O (85:15). The filtrate was fractioned by column chromatography using silica gel and a hexane−EtOAc gradient. Dehydroleucodine (1) was eluted in the hexane−EtOAc (85:15) fraction and recrystallized from EtOAc as a white crystalline solid. Leucodine (2) was isolated from the mother liquid using preparative TLC hexane−EtOAc (70:30) and recrystallized from EtOAc. The identification of these compounds was done by direct comparison with an authentic sample.9 Single-Crystal X-ray Diffraction Analysis and Crystallographic Data for Compound 2. Crystallographic data for all of the structures were deposited in the Cambridge Crystallographic Data Centre, and the deposition number is listed below. The data can be obtained free of charge at www.ccdc.cam.ac.uk (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or [email protected]). Crystallographic data of 2: crystallization from ethyl acetate yielded colorless crystals of 2. X-ray data were collected on a BrukerNonius X8 Proteum CCD diffractometer using Cu Kα radiation. The structures were solved using SHELXT and were refined using SHELXL.26 Crystal data: C15H18O3, Mw = 246.29 g mol−1, space group P212121, a = 7.5528(2) Å, b = 11.7831(3) Å, c = 14.1030(3) Å, α = β = γ = 90.00°, V = 1255.10(5) Å3, T = 90 K, Z = 4, Dc = 1.303 g/ cm3, R1 = 0.0284 (wR2 = 0.0753); Flack parameter = 0.01(4). Deposition number: CCDC 1047867. Cell Lines and Cell Culture. Bone marrow and peripheral blood samples were obtained from human donors with informed consent under Weill Cornell Medical College IRB 0909010629 approval. Mononuclear cells were isolated from the samples using Ficoll-Plaque (Pharmacia Biotech, Piscataway, NY, USA) density gradient separation. Cells were cultured in serum-free medium supplemented with cytokines (50 ng/mL rhFLT-3 ligand, 50 ng/mL rhSCF, 20 ng/ mL rhIL-3, and 20 ng/mL rhIL-6) for 1 h before the addition of drugs. The HL-60 (purchased 9/2010, ATCC), Kasumi-1 (purchased 4/ 2011, ATCC), KG-1 (purchased 9/2010, ATCC), MOLM-13 (kind gift from G. Chiosis-MSKCC 7/2010, 2/2014 authenticated; biosynthesis), MV4-11 (purchased 9/2010, ATCC), THP-1 (purchased 9/ 2010, ATCC), TUR (purchased 1/2010, ATCC), and U937 (purchased 12/2009, ATCC) cell lines were cultured in Iscove’s modified Dulbecco’s medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10−20% fetal bovine serum, according to the culture conditions indicated by ATCC, and were supplemented with 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA) at 37 °C and 5% CO2. Flow Cytometry and Cytotoxicity Assays. The cells were seeded into 96-well plates and were maintained at a concentration of 0.5 million cells per mL. The samples were treated with compounds 1−3 at varying concentrations in triplicate. Cell counting for these experiments was performed using the Invitrogen Cell Countess system with Trypan Blue stain. Compound 3 was obtained from Biomol (Plymouth Meeting, PA, USA). Cell viability was determined after incubating for 48 h. The cells were stained with annexin V-fluorescein isothiocyanate or phycoeritrine and 7-aminoactinomycin (7-AAD) all from Molecular Probes-Invitrogen (Carlsbad, CA, USA) to detect phosphatidylserine exposition and cell permeability, respectively. At least 50 000 events were recorded per condition on either an LSR-II or an LSR-Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA). Data analysis was conducted using FlowJo 9.6 software for Mac OS X (Tree Star, Ashland, OR, USA). Cells negative for annexin V and 7AAD were scored as viable. Analyses and graphs were produced using the GraphPad Prism software (La Jolla, CA, USA). RNA Extraction and Quantitative PCR. MOLM-13 cells were seeded at 0.5 million cells per mL. Cells were treated for 6 h with 20 μM compounds 1−3 and then collected. Total RNA was extracted using the Qiashredder and the Qiagen RNeasy Mini kits (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Real-time PCR was performed using the Taqman RNA-to-CT 1-Step kit (Applied Biosystems, Waltham, MA, USA). The thermal cycling conditions were as follows: one RT step (48 °C, 15 min), one enzyme activation step (95 °C, 10 min), and 40 cycles of denaturation (95 °C,

15 s) and annealing/extension (60 °C, 1 min). Quantitative PCR was performed using probes for HMOX1 (Hs01110251_m1), HSPA1A (Hs00359163_s1), and NFKB1 (Hs00765730_m1). GADPH (Hs02758991_g1) was used as a housekeeping gene, which is an internal control to normalize the variability in the expression levels. All of the probes were provided by Applied Biosystems. Single-plex realtime PCR was performed in triplicate in a StepOne Plus RealTime PCR system (Applied Biosystems), and the PCR products were analyzed with the StepOne Software (Applied Biosystems). The fold changes were calculated using the 2-DDCT method described by Livak and Schmittgen.27 Western Blotting. MOLM-13 cells were seeded at 0.5 million cells per mL and were subjected to treatment with either 10 or 20 μM compound 1 or 3 for 6 h. Cells were lysed in protein lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 5 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM NaF, and Set I protease inhibitor cocktail (EMD Millipore)) on ice for 30 min. Protein lysates were resolved on a 10% SDS-PAGE gel (Invitrogen, Carlsbad, CA, USA) and transferred to an ImmobilonFL polyvinylidene difluoride membrane (EMD Millipore). The membrane was incubated with primary antibodies at 4 °C overnight, then washed and incubated with IRDye 680 goat anti-rabbit or IRDye 800CW goat anti-mouse secondary antibodies (Li-COR, Lincoln, NE, USA) at room temperature for 30 min. The membrane was then washed with phosphate-buffered saline with 0.1% Tween 20X (SigmaAldrich, St. Louis, MO, USA). The membrane was scanned using the Odyssey infrared imaging system (Li-COR). Phospho-NF-κB p65 mouse primary antibody (Cell Signaling, Danvers, MA, USA) and NFκB p65 rabbit primary antibody (Cell Signaling) were used separately to stain the membranes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00383. Crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel (M. L. Guzman): +1 (212) 746-6838. Fax: +1 (212) 7468154. E-mail: [email protected]. *Tel (C. M. Compadre): +1 (501) 686-6493. Fax: +1 (501) 686-6057. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported in part by the University of Arkansas for Medical Sciences College of Pharmacy Research Fund, the Irma T. Hirschl/Monique Weill-Caulier Trust and by the Arkansas Science and Technology Authority, Project No. 15-B-01. P.E.O. is a recipient of a graduate student scholarship from Ecuador’s Higher Education, Science, and Technology Ministry (SENESCYT). M.L.G. is a recipient of the NIH Director’s New Innovator Award Program, 1 DP2 OD00739901. We also thank Dr. V. Morocho of Universidad Técnica Particular de Loja for his help identifying G. verrucosa.

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