Natural products

Amorfrutin C Induces Apoptosis and Inhibits Proliferation in ColonCancer Cells through Targeting MitochondriaChristopher Weidner,†,∥ Morten Rousseau,†,∥ Robert J. Micikas,‡ Cornelius Fischer,† Annabell Plauth,†

Sylvia J. Wowro,† Karsten Siems,§ Gregor Hetterling,§ Magdalena Kliem,† Frank C. Schroeder,‡

and Sascha Sauer*,†,⊥

†Otto Warburg Laboratory, Max Planck Institute for Molecular Genetics, D-14195 Berlin, Germany‡Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853,United States§AnalytiCon Discovery GmbH, D-14473 Potsdam, Germany⊥CU Systems Medicine, University of Wurzburg, D-97080 Wurzburg, Germany

*S Supporting Information

ABSTRACT: A known (1) and a structurally related new natural product(2), both belonging to the amorfrutin benzoic acid class, were isolated fromthe roots of Glycyrrhiza foetida. Compound 1 (amorfrutin B) is an efficientagonist of the nuclear peroxisome proliferator activated receptor (PPAR)gamma and of other PPAR subtypes. Compound 2 (amorfrutin C) showedcomparably lower PPAR activation potential. Amorfrutin C exhibitedstriking antiproliferative effects for human colorectal cancer cells (HT-29and T84), prostate cancer (PC-3), and breast cancer (MCF7) cells (IC50values ranging from 8 to 16 μM in these cancer cell lines). Notably, amorfrutin C (2) showed less potent antiproliferative effectsin primary colon cells. For HT-29 cells, compound 2 induced G0/G1 cell cycle arrest and modulated protein expression of keycell cycle modulators. Amorfrutin C further induced apoptotic events in HT-29 cells, including caspase activation, DNAfragmentation, PARP cleavage, phosphatidylserine externalization, and formation of reactive oxygen species. Mechanistic studiesrevealed that 2 disrupts the mitochondrial integrity by depolarization of the mitochondrial membrane (IC50 0.6 μM) andpermanent opening of the mitochondrial permeability transition pore, leading to increased mitochondrial oxygen consumptionand extracellular acidification. Structure−activity-relationship experiments revealed the carboxylic acid and the hydroxy groupresidues of 2 as fundamental structural requirements for inducing these apoptotic effects. Synergy analyses demonstratedstimulation of the death receptor signaling pathway. Taken together, amorfrutin C (2) represents a promising lead for thedevelopment of anticancer drugs.

Cancer is the third leading cause of death globally,accounting for about 8 million deaths and 13 million

new cases per year.1,2 Colorectal cancer (CRC) is the thirdmost common form of cancer, with 1 million new cases andmore than 600 000 deaths per year.2 Current chemotherapeuticapproaches are hampered by often severe toxic side effects,emergence of drug resistance, and frequent relapse.3 Thediscovery and development of new promising anticancer agentstherefore addresses an urgent need. A large proportion ofanticancer agents in current clinical use are based on naturalproducts or their synthetic analogues,4,5 and purified naturalproducts as well as crude extracts have become a recent focus ofnutrition research aiming to develop functional food andnutraceuticals with significant health benefits.6

We recently reported the characterization of some membersof the amorfrutin family.7,8 These natural products feature aplanar 2-hydroxybenzoic acid core with isoprenyl, benzyl, oralkyl residues and a methoxy or hydroxy group at position C-4.9

Amorfrutins were first extracted from the bastard indigo bushAmorpha f ruticosa;10 however, amorfrutins have also been

found in the roots of Glycyrrhiza foetida Desf. (Leguminosae), aperennial herb endemic to southern Spain (Andalusia) andnorthwest Africa (Morocco and Algeria), as well as in G.acanthocarpa native to Southern Australia.9,11

It recently was reported that amorfrutins A and B(compound 1) strongly reduce insulin resistance and liversteatosis in metabolic mouse models7,8,12 via selective activationof the peroxisome proliferator-activated receptor (PPAR)nuclear receptor class.13 Amorfrutin B (1) was found to bethe most efficient binding molecule of PPARγ among this classof natural products. In addition, amorfrutin A has beenreported to inhibit common anti-inflammatory pathways,14,15 inpart via modulation of PPARγ.16

In the present study, it was shown that amorfrutin C (2) 2-Hydroxy-4-methoxy-3,5-bis(3-methylbut-2-enyl)-6-phenethyl-benzoic acid, strongly inhibited proliferation to kill cancer cells.Interestingly, in contrast to the PPAR agonist 1, compound 2

Received: January 26, 2015

Article

pubs.acs.org/jnp

© XXXX American Chemical Society andAmerican Society of Pharmacognosy A DOI: 10.1021/acs.jnatprod.5b00072

J. Nat. Prod. XXXX, XXX, XXX−XXX

did not efficiently activate PPARγ,8 a nuclear receptor that hasbeen proposed as a target for treating both type 2 diabetes andalso cancer.9 This study further explored the apoptotic effects of2 in HT-29 colon carcinoma cells and provided mechanisticinsights into potential modes of action of 2, which go beyondinteraction with nuclear receptors of the PPAR family.

■ RESULTS AND DISCUSSIONIsolation and Structure Elucidation. Amorfrutin C (2)

was isolated from the roots of G. foetida as a new derivative ofthe known amorfrutin B (1) with an additional prenyl residue.Amorfrutin C gave a molecular formula of C26H32O4 asdeduced by HRMS. The 1H NMR spectrum (Table 1) showedtypical signals of an “AA′BB′C” system of a monosubstitutedphenyl ring. From the COSY and HSQC spectra two prenylgroups and an aromatic methoxy group were deduced. Bothprenyl moieties were found to be directly attached to a fullysubstituted aromatic ring with the aromatic rings linked by aCH2−CH2 bridge. The couplings observed in the HMBCspectrum allowed the positioning of the substituents at the fullysubstituted phenyl ring (Table 1). Particularly, 3JC,H couplingsfrom the methylene groups of the prenyl moieties as well asfrom the methoxy group to C-4 allowed the placement of thelatter between the two prenyl moieties. The other neighbors ofthe prenyl moieties, a hydroxy group and the ethylene chain,were placed by HMBC correlations to C-6 and to C-2,respectively. The substitution of the phenyl ring was completedby a carboxylic acid unit that showed no correlations in theHMBC spectrum.Antiproliferative Activity. Compounds 1 and 2 were

tested for their inhibitory effects on the growth of a small panelof cancer cell lines. HT-29 and T84 colon carcinoma, PC-3prostate cancer, and MCF7 breast cancer cells were treatedwith 1 and 2 for 72 and 96 h, respectively. Cell proliferationwas determined by measurement of cellular DNA content(Table 2). Both amorfrutin compounds inhibited cancer cellproliferation, with compound 2 slightly more potent than 1,with IC50 values of 8 μM (HT-29), 11 μM (T84), 16 μM (PC-3), and 14 μM (MCF7), respectively (Figure 1A), which arelower than the IC50 values observed for cisplatin used as apositive control. Treatment with 1 and 2 above the IC50concentrations resulted in near-complete death of coloncarcinoma cells, whereas the positive controls caused maximally70% and 90% death (Table 2). Importantly, in primary coloncells, 2 showed clearly weaker antiproliferative effects when

compared to colon cancer cells (details are shown in Figure1A).

Compound 2 Triggers Cell-Cycle Arrest and Apopto-sis. Cellular proliferation is generally linked to cell cycleprogression.17 Therefore, the effects of 2 were assessed on keycell cycle modulators by immunoblotting of proteins from HT-29 cells that were treated with 10 μM 2 for 48 h. Compound 2considerably induced the expression of p21/Cip1 and p27/Kip1, two important cyclin-dependent kinase inhibitors.Compound 2 further reduced the expression of cyclins A2,D3, and E2, as well as the cyclin-dependent kinases (CDK) 2,4, and 6 (Figure 1B). Additionally, the effects of 2 on the cellcycle were analyzed by flow cytometry. Compound 2-treatedcells accumulated in the G0/G1 phase (89% vs 66% for 2 vscontrol) with concomitant reduction in the S (2% vs 11%) andthe G2/M phases (7% vs 21%) (Figure 1C). These resultsindicated that the growth inhibitory effect of 2 for HT-29 coloncarcinoma cells was likely a result of G0/G1 cell cycle arrest.In order to shed more light on the cellular effects of 2,

apoptosis was further analyzed in HT-29 cells. In healthy cells,phosphatidylserine (PS) is generally restricted to the innerleaflet of the cell membrane, and the presence of largequantities of PS on the outer leaflet represents a hallmark ofapoptosis.18 Treatment with 20 μM 2 for 48 h increased PSexternalization from 8% to 44% (Figure 2A). Moreover, earlyactivation of caspases (as markers of the signaling network toconnect extrinsic and intrinsic stimuli with downstreamapoptotic events) was measured in HT-29 cells.19 Treatmentwith 20 μM 2 for 2 h significantly induced the enzymaticactivity of the effector caspases 3/7 by 2-fold (Figure 2B).

Chart 1. Chemical Structures of Compounds 1, 2, 2a, and 2b

Table 1. 1H NMR Data (CD3OD, 500 MHz) and 13C NMRData (CD3OD, Data Deduced from HSQC and HMBCSpectra)

proton 2 carbon 2

1 1 109.72 2 142.23 3 125.74 4 161.65 5 121.16 6 160.57 3.13 m 7 32.68 2.72 m 8 37.09 9 142.610 7.14a 10 128.111 7.19a 11 128.112 7.09a 12 125.813 7.19a 13 128.114 7.14a 14 128.1prenyl-1 1′ 3.30 m prenyl-1 1′ 25.2prenyl-1 2′ 4.98 br t (6.5) prenyl-1 2′ 125.0prenyl-1 3′ prenyl-1 3′ 130.9prenyl-1 4′ 1.61 s prenyl-1 4′ 24.9prenyl-1 5′ 1.73 s prenyl-1 5′ 19.9prenyl-2 1″ 3.30 m prenyl-2 1″ 25.2prenyl-2 2″ 5.18 br t (6.5) prenyl-2 2″ 123.2prenyl-2 3″ prenyl-2 3″ 130.9prenyl-2 4″ 1.62 s prenyl-2 4″ 24.9prenyl-2 5″ 1.74 s prenyl-2 5″ 17.0OMe 3.62 s OMe 60.2

COOH b

aAA′BB′C system. bNot observed in HMBC spectrum.

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00072J. Nat. Prod. XXXX, XXX, XXX−XXX

B

Notably, 2 activated caspases 8 and 9 by 3-fold (Figure 2B),indicating activation of the extrinsic (death receptor mediated)as well as the intrinsic (mitochondrial mediated) pathway ofapoptosis. Fluorescence microscopy validated cleavage ofcaspase 3 and further revealed formation of apoptotic bodiesin treated HT-29 cells (Figure 2C). As expected, 2 additionallyinduced significant DNA fragmentation in these cells (Figure2D). Consequently, treatment with 2 also induced cleavage ofthe chromatin-associated poly(ADP-ribose) polymerase(PARP) (Figure 2E); thus 2 efficiently activated the apoptoticcascade in human HT-29 colon carcinoma cells.In contrast to amorfurtin B (1), amorfrutin C (2) appeared

to be only a low-affinity ligand for the peroxisome proliferator-

activated receptors. Competitive binding studies previouslyrevealed affinity values of 9.1, 5.2, and 0.7 μM for PPARα, β/δ,and γ, respectively.8 Since PPARs are also involved in cellgrowth and differentiation,20 their potential role was inves-tigated for the observed antiproliferative and apoptotic effectsof 2. Transfection of short interfering RNA (siRNA) against allthree PPAR subtypes reduced the gene expression of PPARα,β/δ, and γ by 73%, 83%, and 70%, respectively (Figure 3A).However, silencing of the PPARs in HT-29 cells showed noeffects on inhibition of proliferation (Figure 3B) and apoptosis(Figure 3C) induced by compound 2, suggesting a PPAR-independent mechanism. As knockdown of the PPARs was notcomplete, contribution to cellular effects owing to low-affinity

Table 2. Antiproliferative Effects of 1 and 2 in HT-29, T84, PC-3, and MCF7 Cancer Cellsa

HT-29 (colon cancer) T84 (colon cancer) PC-3 (prostate cancer) MCF7 (breast cancer)

IC50 (μM) efficacy (%) IC50 (μM) efficacy (%) IC50 (μM) efficacy (%) IC50 (μM) efficacy (%)

1 20 ± 1 98 ± 4 13 ± 1 99 ± 3 32 ± 3 95 ± 5 33 ± 1 94 ± 32 8.1 ± 0.5 95 ± 3 11 ± 1 99 ± 3 16 ± 2 95 ± 5 14 ± 1 92 ± 3cisplatin 11 ± 2 91 ± 6 15 ± 1 87 ± 3 131 ± 19 n.d. >100 ± n.d. n.d.oxaliplatin 1.6 ± 0.3 70 ± 2 1.1 ± 0.3 87 ± 4 0.8 ± 0.1 87 ± 2 0.4 ± 0.0 83 ± 25-FU 5.3 ± 1.2 68 ± 5 6.2 ± 10.7 76 ± 18 4.9 ± 0.4 80 ± 2 0.9 ± 0.1 80 ± 2irinotecan 1.9 ± 1.8 71 ± 21 1.2 ± 0.4 82 ± 5 0.8 ± 0.1 76 ± 2 0.9 ± 0.3 82 ± 4etoposide 2.2 ± 0.3 97 ± 62 3.0 ± 0.3 90 ± 3 1.1 ± 0.3 84 ± 4 0.8 ± 0.2 78 ± 3

aEfficiency is the maximal observed induction of cell death after treatment relative to nontreated cells (set to 0%). HT-29 and PC-3 cells weretreated for 72 h; MCF7 and T84 cells for 96 h. Treatment times were adjusted according to cell-specific variation in proliferation. n.d., notdetermined.

Figure 1. Antiproliferative activity of compound 2 in human cancer cell lines. (A) HT-29 and T84 colon carcinoma, PC-3 prostate cancer, MCF7breast cancer, and CCD 841 CoN primary colon cells were treated with concentration series of 2 for 72 h (HT-29 and PC-3) and 96 h (T84, MCF7,and CCD 841 CoN), respectively. The relative number of cells was determined. Notably, in CCD 841 CoN primary colon cells, compound 2(amorfrutin C) showed lower antiproliferative activity than in colon cancer cells. In primary colon cells, an IC50 value of ∼49 μM (and an efficacy of∼87%) was determined, and in HT-29 colon cancer cells the IC50 value was ∼8 μM (and an efficacy of ∼96%). Data are expressed as means ± SD (n= 4). (B) Whole cell lysates were analyzed for the expression of the cell cycle regulating proteins p21, p27, cyclin A2, cyclin D3, cyclin E2, CDK2,CDK4, CDK6, and GAPDH. (C) Cell cycle analysis of HT-29 cells. Histograms (left) show one representative experiment for each treatmentcondition. Bar plots (right) show percentages of the cell population in apoptotic SubG1, G0/G1, S, and G2/M phases of the cell cycle and areexpressed as means ± SEM (n = 3). *p ≤ 0.05, ***p ≤ 0.001 vs control.

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00072J. Nat. Prod. XXXX, XXX, XXX−XXX

C

binding of 2 to PPARs remains possible. However, in generalactivation of PPARs by high-affinity ligands such as amorfrutinsA and B usually does not result in strong antiproliferativeeffects, as was shown recently.7,8

Global Gene Expression Analysis. In order to shed morelight on the potential mechanism of action of 2, thetranscriptome of HT-29 cells that were treated with 30 μM 2for 4 h was analyzed using RNA sequencing. Noteworthy, 409genes were highly regulated after short-term treatment (80 up-,329 downregulated; Supporting Information Excel file 1).These genes were further subjected to gene ontology (GO)overrepresentation analysis, and an integrative network wasbuilt using Cytoscape21 (Figure S1, Supporting Information).As expected, GO terms such as “proliferation”, “cell cycle”, “celldeath”, or “apoptosis” were highly enriched in the regulatedgenes (Figure 4, Supporting Information Excel file 2).Strikingly, “oxidative stress” as well as “mitochondria” relatedGO terms were further significantly enriched in the tran-

scriptome of these cells (Figure 4), indicating a mechanisticrole of reactive oxygen species (ROS) and intrinsic apoptosispathways for the effects of compound 2.

ROS Formation Is Not Causal for Antiproliferative andApoptotic Effects of Compound 2. Cancer cells aregenerally characterized by an imbalance of ROS, and increasingoxidative stress can activate apoptotic pathways.22 It was nextasked if compound 2 leads to formation of ROS in HT-29 cells.To measure intracellular ROS formation during treatment with2, the fluorescence of chloromethyl dichlorofluorescein (CM-DCF) was detected in living HT-29 cells. Of note, 30 μM 2induced significant accumulation of ROS during the first hoursof treatment (Figure 5A). Co-treatment with commonantioxidants prevented completely the formation of ROS inthese cells (Figure 5A). However, the antioxidants did notrescue the cancer cells either from inhibition of proliferation(Figure 5B) or from apoptosis (Figure 5C) induced by 2.

Figure 2. Activation of apoptosis in HT-29 colon carcinoma cells by compound 2. (A) Phosphatidylserine externalization of HT-29 cells wasdetermined by flow cytometry of annexin V-FLUOS- and propidium iodide (PI)-stained cells. Scatter plots (left) show one representativeexperiment for each treatment condition. Bar plots (right) show percentage of cell populations in apoptosis, comprising early (annexin positive, PInegative) and late stage (annexin positive, PI positive) apoptotic events. Bars represent means ± SEM (n = 3). (B) HT-29 cells were treated with 30μM 2 for 2 h. Enzymatic activation of caspases 2, 3/7, 6, 8, and 9 was determined by luminescent assays. Data are normalized to control treatmentand are expressed as means ± SEM (n = 4). (C) HT-29 cells were treated with 30 μM 2 for 6 h. Cleavage of caspase 3 was visualized by fluorescencemicroscopy. Nuclei were stained with DAPI. Apoptotic bodies are marked with white arrows. (D) Effects of 2 on DNA fragmentation. Accumulationof DNA in the cytosol was determined by detection of BrdU-labeled DNA (left) and oligonucleosomes (right) using ELISA. Data are normalized tothe control treatment and are expressed as means ± SEM (n = 4). (E) HT-29 cells were treated with 10 μM 2 for 48 h. Whole cell lysates wereanalyzed for protein expression of total and cleaved PARP. n.s. not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 vs control.

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00072J. Nat. Prod. XXXX, XXX, XXX−XXX

D

These results suggest that the formation of ROS might not be(directly) causal for the cellular effects of compound 2.Compound 2 Disrupts Mitochondrial Integrity in HT-

29 Colon Carcinoma Cells. Mitochondria not only areindispensable for cellular energy production and metabolismbut are also regulators of the intrinsic pathway of apoptosisleading to cell death.23 Considering the observed activation ofcaspase 9 (Figure 2B) and the mitochondria-related geneexpression changes (Figure 4), it was assumed that compound2 could potentially induce mitochondrial dysfunction.In healthy cells, the inner mitochondrial membrane, nearly

impermeable to all ions, contributes to the robust formation ofan electrochemical gradient leading to the mitochondrialtransmembrane potential (ΔΨm) required for ATP synthesis.A long-lasting opening of the mitochondrial permeabilitytransition pore (MPTP) results in permanent ΔΨm dissipationand cell death.23 Cancer cells exhibit increased ΔΨm due toaccelerated metabolism, making test compounds that selectivelyfacilitate mitochondrial membrane permeabilization interestingfor drug development.24,25 Notably, treatment of compound 2in HT-29 cells led to potent dissipation of the ΔΨm with anIC50 value of 0.6 μM within a few minutes (Figure 6A).The effects on the MPTP were further tested by flow

cytometry. Compound 2 induced a permanent opening of theMPTP in these cells (Figure 6B,C). In contrast, MPTP opening

was partly prevented by preincubation with cyclosporine A(CsA), an inhibitor of cyclophillin D that is a component of theMPTP.25 However, preincubation with CsA was not sufficientfor preventing ΔΨm dissipation (Figure 6D) or for preventingthe apoptotic effects of 2 (Figure 6E) (actually, CsA potentiatesthe cytotoxicity of 2). These observations suggest that ΔΨmdissipation and apoptosis are not direct consequences of theMPTP opening induced by 2. Instead, compound 2 may inducemitochondrial membrane depolarization by other mechanisms,e.g., protonophoric uncoupling.In general, perturbed mitochondria should display functional

alterations. To investigate mitochondrial oxygen consumptionin living HT-29 cells during treatment with compound 2, thefluorescence of an oxygen-sensitive dye was measured.Strikingly, treatment with 2 and 10 μM of compound 2resulted in elevated mitochondrial oxygen consumption similarto the protonophoric uncoupler carbonyl cyanide 3-chlorophe-nylhydrazone (CCCP). In contrast, inhibition of the electrontransport chain at complex III by antimycin A (AMA) blockedoxygen consumption (Figure 6F,G).Generally, mitochondrial perturbations induce glycolysis to

compensate for reduced ATP synthesis. Since the pyruvate-to-lactate conversion is the main contributor to extracellularacidification in unsealed cellular systems (in balance withconstant levels of atmospheric carbon dioxide), the pH is anindicator for glycolytic flux.26 Therefore, extracellular acid-ification was measured by fluorescence quenching of a pH-sensitive dye. Of note, treatment with 2 and 10 μM 2 resultedin 3- and 6-fold elevated extracellular acidification rates(ECAR) with 1.5 and 2.9 × 10−9 [H+]/min, respectively.Similarly, the mitochondrial perturbators CCCP and AMA alsoinduced extracellular acidification 3- and 7-fold, respectively(Figure 6H,I). Overall, these results demonstrate thatcompound 2 induced mitochondrial damage in HT-29 coloncarcinoma cells, leading to structural and metabolic dysfunctionand finally to apoptosis.In order to shed more light on structure−activity relation-

ships, compound 2 was chemically modified by blocking the C-1 carboxylic acid and the C-2 hydroxy groups. Both residuescan act as hydrogen bond donors, showing acid dissociationconstants (pKa) of 3.5 and 11.6, respectively (Figure S2,Supporting Information). However, in nonpolar environments(such as biological membranes) acid dissociation constants of

Figure 3. Role of peroxisome proliferator-activated receptors (PPARs) for antiproliferative and apoptotic effects of compound 2. (A) HT-29 cellswere transfected with nonselective siRNA or with an equimolar mixture of PPAR-selective siRNA (against PPAR α, β/δ, and γ) for 48 h. RNAireduced the gene expression of PPAR α, β/δ, and γ by 74%, 83%, and 70%, respectively. Data are expressed as means ± SEM (n = 4). (B) HT-29cells were transfected as in part A and treated subsequently with 20 μM 2 for an additional 48 h. The relative number of cells was then determined.Data are expressed as means ± SD (n = 8). (C) HT-29 cells were transfected as in part A and subsequently treated with 30 μM 2 for an additional 24h. Phosphatidylserine externalization was determined as described in Figure 2. Bar plots (right) show percent of cell population in apoptosis (means± SEM, n = 6). n.s. not significant, ***p ≤ 0.001 between nonselective and PPAR-selective siRNA.

Figure 4. Global gene expression analysis of HT-29 cells treated withcompound 2. Cells were treated for 4 h, and isolated RNA wassequenced subsequently. Expressed RNA transcripts were thenglobally analyzed for overrepresented gene ontology (GO) termsthat were subjected to network analyses. Prominent network clusterswere selected and shown with adjusted p values for these terms. Dataare expressed as medians ± range.

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00072J. Nat. Prod. XXXX, XXX, XXX−XXX

E

compounds can shift by up to 7 units.27 Therefore, the 1-carboxy and 2-hydroxy methylated synthetic derivatives 2a and2b were investigated, respectively. Methylation of either ofthese functional groups was sufficient to completely abolishmitochondrial membrane depolarization (Figure 7A) andapoptosis (Figure 7B) in HT-29 cells. These observationsindicate that the 2-hydroxybenzoic acid motif in compound 2 isa relevant feature, potentially by promoting proton transportthrough the inner mitochondrial membrane, leading to ΔΨmdissipation. Salicylic acid derivatives such as 2 are significantlymore acidic than their corresponding benzoic acid analogues(2a). In conjunction with the compound’s high lipophilicity,the acidity of 2 might stimulate protonophoric mitochondrialuncoupling, possibly by transfer of protons over the innermitochondrial membrane.Additive and Synergistic Effects of Compound 2.

Simultaneous treatment with compounds that make use ofdifferent molecular mechanisms might produce additive orsynergistic effects on cancer cell proliferation and death.28

Compound 2 was tested in combination with various anticanceragents for inhibition of proliferation and apoptosis in HT-29cells using the Loewe additivity model (see ExperimentalSection). Interestingly, combinations with the DNA-cross-linking drug cisplatin (Figure 8A) as well as with thetopoisomerase I inhibitor irinotecan (Figure 8B) achievedadditive effects on proliferation inhibition. These data suggestoverlapping molecular mechanisms resulting in antiproliferativeeffects. However, for all cancer cell lines tested in this study,compound 2 achieved higher maximal cell growth arrestefficiency than cisplatin and irinotecan (Table 2), indicating the

existence of additional molecular events. Potential synergy onapoptotic events was evaluated by analyzing simultaneousactivation of the alpha Fas receptor ligand (αFAS) and theTNF-related apoptosis inducing ligand (TRAIL). Synergy wasassessed with the Bliss independence model.29 Strikingly, 2synergistically induced PS externalization with αFAS andTRAIL, suggesting that 2 does not bind to death receptors.However, since compound 2 also activated caspase 8 (Figure2B), it probably activated the death receptor signaling pathwaydownstream of the death receptors, as recently described formitochondrial perturbators.30

Conclusion. In summary, amorfrutin C (2), isolated fromthe roots of G. foetida, showed potent antiproliferative effects indifferent cancer cell lines by triggering cell cycle arrest andinducing apoptosis. Mechanistically, the effects of 2 could becorrelated with mitochondrial depolarization, although furtherdetailed analysis is needed to identify potentially involvedcellular targets. Synergy studies and extrinsic apoptosisactivation further indicated stimulation of the death receptorsignaling pathway.Since cancer cell mitochondria are structurally and function-

ally different from their normal counterparts, cancer cells aremore susceptible to mitochondrial toxicity than normal cells.31

Thus, targeting mitochondria function appears to be apromising strategy for cancer therapy.25 In line with thisconceptual framework, the molecular interference of amorfrutinC (2) with mitochondrial function seems to produce strongerantiproliferative effects in colon cancer cells than in normalcolon cells (Figure 1A). On the other hand, mechanisticallyinteresting but lipophilic natural products such as 2 require

Figure 5. Role of reactive oxygen species (ROS) in antiproliferative and apoptotic effects of compound 2. (A) HT-29 cells were incubated with 30μM 2 in the absence or presence of the antioxidants N-acetylcysteine (NAC, 1 mM), glutathione (GSH, 2.5 mM), 3H-1,2-dithiole-3-thione (D3T,25 μM), α-tocopherol (αTOC, 50 μM), and ascorbic acid (AA, 500 μM). Intracellular ROS were kinetically detected (left) by use of CM-H2DCFDAin living cells. Bar plots (right) show the area under the curve (AUC). Data are expressed as means ± SEM (n = 7). (B) HT-29 cells were treatedwith concentration series of compound 2 in the absence or presence of antioxidants for 48 h. For measurement of antiproliferative effects, the relativenumber of cells was determined by use of a DNA-binding fluorescent dye. Data are expressed as means ± SD (n = 4). (C) HT-29 cells were treatedwith 30 μM 2 in the absence or presence of antioxidants for 24 h. Apoptosis was determined as described above. Bars represent means ± SEM (n =3). ***p ≤ 0.001 vs control, n.s. not significant vs compound 2-only treated cells; ###p ≤ 0.001 vs compound 2-only treated cells.

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00072J. Nat. Prod. XXXX, XXX, XXX−XXX

F

chemical optimization and/or the use of molecular carriers forefficient application in vivo. Further systematic safety andpharmacokinetic studies of the novel amorfrutin C (2) andanalogues will be important to develop potent candidates forpreclinical trials. Notably, amorfrutin B (1) has been extensivelyanalyzed in in vivo models, and no toxic side effects wereobserved.8 Taken together, these data show that amorfrutin C(2) and natural or synthetic analogues thereof may becomepromising candidates for developing anticancer drugs.

■ EXPERIMENTAL SECTION

General Experimental Procedures. Chemicals were purchasedfrom the following sources: staurosporine, irinotecan, cisplatin, andantimycin A from LKT Laboratories (Biomol, Hamburg, Germany);oxaliplatin from Cayman Chemical (Biomol). Paclitaxel, 5-fluorouracil,etoposide, doxorubicin, N-acetylcysteine (NAC), glutathione (GSH),3H-1,2-dithiole-3-thione (D3T), α-tocopherol (αTOC), ascorbic acid(AA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), andcyclosporine A (CsA) were purchased from Sigma-Aldrich (Tauf-

Figure 6. Mitochondrial dysfunction induced by compound 2. (A) Loss of mitochondrial transmembrane potential (ΔΨm) detected by fluorometry.HT-29 cells were incubated with concentration series of 2 for 5 min. Data are expressed as means ± SD (n = 4). (B) Opening of the mitochondrialpermeability transition pore (MPTP) determined by calcein/CoCl2 fluorescence. HT-29 cells were treated for 30 min with 2 in the absence orpresence of cyclosporin A (CsA, 4 μM). Histograms show one representative experiment for each treatment condition. (C) Mean of calcein/CoCl2fluorescence intensities. Bars show means ± SEM (n = 6). (D) Effect of MPTP inhibition on compound 2-induced ΔΨm dissipation. Note thatblocking the pore with CsA during pretreatment led to hyperpolarization of the mitochondrial membrane. Data are expressed as means ± SD (n =6). (E) Effect of MPTP inhibition on compound 2-induced cell death. HT-29 cells were treated with 30 μM 2 in the absence or presence of CsA (2μM) for 24 h. Apoptosis and necrosis was determined by flow cytometry (means ± SEM, n = 3). (F) Mitochondrial oxygen consumptiondetermined by fluorescence quenching. Fluorescence lifetime increased with reduction in extracellular oxygen concentration. Data are expressed asmeans ± SEM (n = 8). AMA, antimycin A; CCCP, carbonyl cyanide 3-chlorophenylhydrazone. (G) The rate of fluorescence lifetime change wasdetermined between 20 and 60 min of the treatment shown here in F and plotted relative to untreated cells, giving the relative oxygen consumption.Data are expressed as means ± SEM (n = 8). (H) Extracellular acidification determined by fluorescence quenching. HT-29 cells were labeled with apH-sensitive probe and treated again with the compounds indicated. Fluorescence lifetime was transformed to pH values. (I) Extracellularacidification rate (ECAR) was determined between 20 and 150 min of treatment H and plotted as hydrogen ion concentration change per minute.Data are expressed as means ± SEM (n = 8). n.s. not significant, **p ≤ 0.01, ***p ≤ 0.001 vs untreated cells; #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001 vscompound 2-only treated cells.

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00072J. Nat. Prod. XXXX, XXX, XXX−XXX

G

kirchen, Germany). Human αFAS (clone CH11, #05-201) and TRAIL(#GF092) were purchased from Merck (Darmstadt, Germany).Compound 1 was isolated from the fruits of Amorpha f ruticosa andanalyzed in detail as described recently.8,12 NMR spectra wereacquired on a Bruker 500 MHz and a Varian INOVA 600 MHzspectrometer. ESIMS were recorded on an API 165 LC/MS massspectrometer coupled with an Agilent 1100 HPLC. Preparative HPLCwas performed with a modified Sepbox system (Sepiatec). For furtherinformation we refer to Supporting Information - Additionalinformation for the Experimental Section.Plant Material. Roots of Glycyrrhiza foetida were collected in

Tiflet, Morocco, in June 2008 by Thomas Friedrich, Friedrich NatureDiscovery (FND, http://www.friedrichnaturediscovery.de/en/index.html). AnalytiCon Discovery further processed the roots forcompound extraction. AnalytiCon Discovery has neither a directcontract with Morocco nor a permit from the Moroccan authoritiesbut instead purchased the plant material from FND. A voucherspecimen with the number ACD-V-20720 is deposited at AnalytiConDiscovery. FND exports several medicinal plants from Morocco toGermany and other countries in the world via the Moroccan-basedcompany Sahara Exporters sarl (http://www.saharaexporters.com/en/index.html). In Analyticon’s contract with FND, it is stipulated that itis the responsibility of FND to obtain all necessary collection andexport permits. FND has an export permit for all plants in the portfoliobut no single permit for the collection of G. fetida used in this study. Atthe time of collection in 2008, before the Nagoya protocol came intoforce and worldwide practice, this was neither necessary nor applicableowing to a lack of fully established legal and administrativeframeworks.

Extraction and Isolation. The procedure for compound 1 wasdescribed recently8 whereas compound 2 was mentioned in a previouspaper but so far never substantially characterized.8 In this study, toisolate compound 2 (2-Hydroxy-4-methoxy-3,5-bis(3-methylbut-2-enyl)-6-phenethylbenzoic acid), 1 kg of air-dried and powderedroots was extracted twice with methyl tert-butyl ether (46.3 g). MPLCfractionation (RP-18, 250 × 50 mm) with a gradient from 50%methanol to 100% methanol yielded five fractions containingresorcinol derivatives. Further separation by preparative HPLC (SelectB 10 μm, 250 × 50 mm, flow rate 80 mL/min, gradient with CH3OH/ammonium formate buffer adjusted with formic acid to pH 4.0)yielded pure compound 2 (22 mg).

Amorfrutin C (2): yellow powder; 1H and 13C NMR data, see Table1; (+)-ESIMS m/z 409 [M + H]+, 391 [M − H2O + H]+, 335;(−)-ESIMS 407 [M − H]−; (−)-HRESIMS m/z 407.2221 [M − H]−

(calcd for C26H31O4 407.2221).Compounds 1 and 2 are available as from Analyticon Discovery,

product numbers NP-15142 and NP-15934, respectively. A number of

Figure 7. Role of carboxylic acid and hydroxy group residues ofcompound 2 on mitochondrial transmembrane potential (ΔΨm) andapoptosis induction. (A) Loss of mitochondrial transmembranepotential (ΔΨm) detected by fluorometry. HT-29 cells were incubatedfor 5 min with a concentration series of the compounds indicated andlabeled with the mitochondria-selective JC-1 dye. Data are expressedas means ± SD (n = 4). (B) HT-29 cells were treated with thecompounds indicated for 24 h. Apoptosis was determined by flowcytometry. Bars represent means ± SEM (n = 4). n.s. not significant,***p ≤ 0.001 vs control; ###p ≤ 0.001 vs compound 2.

Figure 8. Additive and synergistic effects of compound 2. (A)Isobolographic analysis of antiproliferative effects for compound 2 andirinotecan. HT-29 cells were treated with combinations of compound2 and irinotecan for 72 h. The relative number of cells was determinedas described above. IC70 data are expressed as means ± SD (n = 4). CI,combination index. (B) Isobolographic analysis for antiproliferativeeffects of 2 and cisplatin. (C) Synergistic apoptosis induction of 2 withαFAS and TRAIL. HT-29 cells were treated with 2 (10 μM), αFAS(10 ng/mL), TRAIL (30 ng/mL), or combinations thereof for 48 h.Apoptosis was determined by flow cytometry. Expected combinationeffects were calculated according to the Bliss independence model andassumed additivity on apoptosis induction. Bars represent means ±SEM (n = 4). **p ≤ 0.01, ***p ≤ 0.001 vs untreated cells; ###p ≤0.001 between observed and expected combination effects.

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00072J. Nat. Prod. XXXX, XXX, XXX−XXX

H

amorfrutin-related natural products, which were tested in pilotexperiments and which displayed similar properties to amorfrutin C,were obtained from Analyticon Discovery, namely, product numbers2-Hydroxy-4-methoxy-3,5-bis(3-methylbut-2-enyl)-6-pentylbenzoicacid (3), 2,4-Dihydroxy-3,5-bis(3-methylbut-2-enyl)-6-pentylbenzoicacid (4), 2,4-Bis(3-methylbut-2-enyl)-5-pentylbenzene-1,3-diol (5), 2-(2-Hydroxy-3-methylbut-3-enyl)-4-(3-methylbut-2-enyl)-5-pentylben-zene-1,3-diol (6), and 2-(2-Hydroxy-3-methylbut-3-enyl)-4-(3-meth-ylbut-2-enyl)-5-phenethylbenzene-1,3-diol (7) (see Supporting In-formation, 1H NMR spectra for 2−7 and LC-MS spectra for 2−7).Preparation of Derivatives 2a and 2b. Compound 2 (100 mg)

was dissolved in anhydrous acetone (5 mL) under argon. Potassiumcarbonate (339 mg, 10 equiv) was added to the stirring solution, andthe resulting suspension was then cooled to 0 °C. To the chilledsuspension was added methyl iodide (35 mg, 2 equiv), and thereaction was immediately allowed to warm to room temperature. Thereaction was stirred at room temperature for 4 h, followed byneutralization at 0 °C using 1 M HCl. The acidic solution wasextracted using diethyl ether and hexanes (1:1, 3 × 1 mL), and thecombined organic collection was washed with H2O (5 mL). Theorganic phase was dried over Na2SO4 and concentrated under reducedpressure, yielding intermediate 2c as a yellow oil (as indicated inSupporting Information, Experimental Section). Without furtherpurification, intermediate 2c was transferred using DMSO (0.3 mL)to a solution of potassium hydroxide (249 mg, 20 equiv) in 5%aqueous DMSO (5 mL). The basic solution was heated to 120 °C for8 h under argon in a sealed container. Subsequently, the reaction wascooled to 0 °C and acidified with 1 M HCl. The acidic solution wasextracted using diethyl ether (3 × 20 mL), and the combined organiccollection was dried over Na2SO4 and concentrated under vacuum.Column chromatography (silica gel; hexanes/EtOAc = 10:1progressing to 2:3) afforded acid 2a (44 mg, 47% yield) as a colorlessoil: 1H NMR (600 MHz, CDCl3) δ 7.29−7.15 (5H, m,), 5.24 (1H, m),5.08 (1H, m), 3.84 (3H, s), 3.73 (3H, s), 3.40 (4H, d), 2.99−2.81 (4H,m), 1.79 (3H, br s), 1.76 (3H, br s), 1.71 (3H, br s), 1.69 (3H, br s);13C NMR (150 MHz, CDCl3) δ 172.4, 159.4, 155.0, 141.9, 138.1,131.82, 131.76, 130.6, 128.4, 128.3, 127.1, 126.0, 124.0, 123.6, 123.062.8, 61.5, 37.4, 33.0, 25.7, 25.6, 25.5, 23.7, 18.2, 18.0; (+)-HRESIMSm/z 445.23395 [M + Na]+ (calcd for C27H34NaO4

+, 445.23493). Ascheme of the synthesis strategy is outlined in Supporting Information(Experimental Section).Compound 2b was obtained as a byproduct of methylation of

compound 2 and isolated by column chromatography (silica gel;hexanes/ethyl acetate = 10:1 progressing to 2:3). 1H NMR (600 MHz,CDCl3) δ 7.33−7.27 (2H, m), 7.24−7.18 (3H, m), 5.26 (1H, m), 5.04(1H, m), 3.98 (3H, s), 3.73 (3H, s), 3.42−3.37 (4H, m), 3.20−3.16(2H, m), 2.82−2.77 (2H, m), 1.80 (3H, br s), 1.74 (3H, br s), 1.70(3H, br s), 1.68 (3H, br s); 13C NMR (125 MHz, CDCl3) 172.1,161.5, 160.2, 142.3, 141.4, 131.8, 131.5, 128.4, 128.1, 126.2, 126.0,124.2, 122.8, 121.2, 109.5, 61.5, 52.3, 37.6, 33.2, 25.7, 25.6, 25.3, 23.5,18.1, 18.0; (+)-HRESIMS m/z 423.25220 [M + H]+ (calcd forC27H35O4

+, 423.25299).Standard Biological Procedures. Standard procedures such as

cell culturing, cell cycle analysis, immuno-(Western) blotting, andcaspase activation assay are summarized in the Supporting Information(for the Experimental Section).pKa Determination. Acid dissociation constants were measured at

Sirius Analytical (Forrest Row, UK). In brief, compound 2 was titratedfrom pH 12.0−2.0 at concentrations of 32−26 μM under methanol/water cosolvent conditions and from pH 12.5 to 8.9 at concentrationsof 30−21 μM under aqueous conditions, respectively. Titration wasperformed in the presence of 0.15 M KCl at 25 °C. Analysis was doneusing the UV metric method.Proliferation Assay. To investigate the growth arrest of HT-29,

T84, PC-3, and MCF7 cancer cells and normal CCD 841 CoN cells,the cells were seeded in black 384-well plates (#3712, Corning, FisherScientific, Schwerte, Germany) with a density of 750 cells/well (HT-29), 250 cells/well (PC-3), and 900 cells/well (T84, MCF7, and CCD841 CoN), respectively. One day later, the cells were treated withconcentration series of the tested compound, as indicated for 72 h

(HT-29 and PC-3) or 96 h (T84, MCF7, and CCD 841 CoN),respectively. For cotreatment of HT-29 cells with antioxidants, thetreatment time was reduced to 48 h. The relative number of cells wasdetermined by measurement of cellular DNA content applying thefluorescent CyQUANT NF cell proliferation assay kit (LifeTechnologies), according to the manufacturer’s instructions. Fluo-rescence intensity was measured with the POLARstar Omega (BMGLabtech, Ortenberg, Germany) and was transformed to relative cellnumber. For concentration series, data were fitted using GraphPadPrism 5.0 according to the equation Y = Top + (Bottom − Top)/(1 +10(LogIC50−X) × Hill slope) with variable Hill slope. Efficiency is themaximal observed induction of inhibition (100% − Bottom) relativeto nontreated cells (set to 0%). IC50 is the concentration that isrequired for 50% inhibition related to the compound’s specific(maximal) efficiency, which often is not sufficient to inhibit 50% of allpresent cells. For a better comparison of proliferation inhibition todetermine additive effects, the IC70 value was calculated as theconcentration required for 70% inhibition related to the total numberof cells. Additive effects were determined by treatment with compoundmixtures with the following ratios: 7:0, 6:1, 5:2, 4:3, 3:4, 2:5, 1:6, and0:7. HT-29 cells were treated with different concentration series ofthese compound mixtures, and IC70 data were calculated and plottedas isobolograms according to the Loewe additivity model.32,33 Thecombination index (CI) for compounds x and y in each mixture (M)was calculated as follows: CI(M) = IC70(Mx)/IC70(x) + IC70(My)/IC70(y).

Annexin V Assay. Phosphatidylserine externalization was analyzedusing the annexin-V-FLUOS staining kit (Roche Diagnostics,Mannheim, Germany), according to the manufacturer’s instructions.Flow cytometry was performed using an Accuri C6 apparatus (BDBiosciences, Heidelberg, Germany), and data were analyzed withFlowJo 7.6 (Tree Star). Apoptosis was defined as annexin positive/PInegative and annexin positive/PI positive, whereas necrosis wasdefined as annexin negative/PI positive. Synergistic effects wereinvestigated by use of the Bliss independence model,29 which isdefined by the equation Exy = Ex + Ey − ExEy, where E is the fraction ofcells in apoptosis and Exy is the (expected) additive effect of drugs xand y as predicted by their individual effects Ex and Ey.

DNA Fragmentation Assays. Accumulation of DNA in thecytoplasm of treated HT-29 cells was determined by use of theCellular DNA Fragmentation ELISA and the Cell Death DetectionELISA kits (both Roche Diagnostics). For the Cellular DNAFragmentation ELISA, HT-29 cells were seeded in 96-well plates(TPP, Biochrom, Berlin, Germany) with a density of 13 000 cells/wellin the presence of 10 μM 5-bromo-2′-deoxyuridine (BrdU) andincubated for 2 days at 37 °C. After removal of unincorporated BrdU,cells were treated for 4 h. The cytosolic fractions were harvested andanalyzed on a 96-well half-area clear high-binding microplate (#3690,Corning), according to the manufacturer’s instructions. For the CellDeath Detection ELISA, HT-29 cells were seeded in 96-well plates(TPP) with a density of 20 000 cells/well and incubated for 1 day at37 °C. Cells were then treated for 4 h. The cytosolic fractions wereharvested and analyzed according to the manufacturer’s instructions.Absorbance was measured using the POLARstar Omega (BMGLabtech). Cell-free samples were used as background control forsubtraction, and data were normalized to vehicle-treated cells.

Fluorescence Microscopy. For visualization of cleaved caspase 3,HT-29 cells were seeded on 13 mm coverslips (Sarstedt, Nurnbrecht,Germany) and placed in 24-well plates (Nunc, Wiesbaden, Germany)with a density of 125 000 cells/well, 1 day before treatment. Treatedcells were washed with PBS, fixed with 4% formaldehyde/PBS for 15min, permeabilized with 0.3% Triton X-100/PBS (PBS-T) for 10 min,and subsequently blocked with 5% goat serum (Sigma-Aldrich) inPBS-T (0.3%) for 60 min (at room temperature each). Cells were thenincubated with primary antibody against cleaved caspase 3 (#9664,Cell Signaling Technology, Merck, Darmstadt, Germany) diluted(1:200) in 1% BSA/PBST (0.3%) at 4 °C overnight. Cells werewashed in PBS-T (0.3%) labeled with anti-rabbit IgG (H+L) andF(ab′)2 fragment Alexa Fluor 488 conjugate (#4409, Cell SignalingTechnology) diluted (1:1000) in 1% BSA/PBS-T (0.3%) at room

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00072J. Nat. Prod. XXXX, XXX, XXX−XXX

I

temperature for 1 h. Finally, coverslips were counterstained withProLong Gold Antifade Mountant solution (containing DAPI) (LifeTechnologies) and incubated at room temperature for 24 h.Fluorescence microscopy was performed on the LSM700 (Zeiss,Jena, Germany).RNA Interference (RNAi). HT-29 cells were transfected with an

equimolar mixture of Silencer Select Validated siRNA against PPAR α,β/δ, and γ (10 nM each) or 30 nM nonspecific Negative ControlsiRNA (all Life Technologies) using the Trans-IT TKO transfectionreagent (Mirus Bio, VWR, Darmstadt, Germany), according to themanufacturer’s instructions. Transfection reagent/siRNA complexeswere prepared in opti-MEM (Life Technologies), incubated at roomtemperature for 30 min, and added to the HT-29 cell suspension inantibiotic-free DMEM/F12/FBS directly before seeding. Cells wereseeded with a density of 850 cells/well (384-well plate) and 150 000cells/well (12-well plate) for proliferation and annexin V assays,respectively. After 48 h, the cells were harvested for gene expressionanalysis or additionally treated by adding 3-fold concentratedcompound or vehicle control. Since the cells that were treated withDMSO as vehicle control did not show any growth-inhibiting effects,the total incubation time for subsequent experiments with these cellshad to be reduced to prevent confluence. A pool of siRNAs against allPPAR subtypes was used to prevent any potential compensatoryeffects. Cells were analyzed for proliferation and annexin V after 48and 24 h of compound treatment, respectively.Gene Expression Analysis. Expression of PPARs was analyzed by

quantitative real-time PCR, as described recently.34 Global RNAexpression was analyzed by RNA sequencing using four biologicalreplicates for each sample. A 1 μg amount of total RNA was used togenerate the cDNA library by use of the TruSeq RNA Sample Prep kitv2 (Illumina, San Diego, CA, USA) according to the manufacturer’sinstructions. The libraries were sequenced using HiSeq 2500(Illumina) in paired-end-50nt mode. Sequencing quality was assessedusing FastQC.35 Sequence reads data were deposited at the EuropeanNucleotide Archive (accession number: PRJEB7551), Web-linkhttp://www.ebi.ac.uk/ena/data/view/PRJEB7551. Reads were map-ped using STAR,36 and quantification of reads mapping to genefeatures (Gencode 16) was done using HTSeq.37 Differentiallyexpressed genes were defined as those that differed by more than 4-fold with corrected p-value ≤ 0.0001 and were identified usingedgeR.38 Differentially expressed genes were analyzed using Cyto-scape21 and BiNGO.39

Detection of Reactive Oxygen Species. Formation of intra-cellular ROS was detected with the ROS-sensitive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Life Technologies) according to the manufacturer’sinstructions. One day before treatment HT-29 cells were seeded in96-well plates (TPP) with a density of 15 000 cells/well. Beforetreatment, adherent cells were washed with prewarmed PBS andloaded with 50 μM dye diluted in PBS. Cells were then incubated at 37°C for 30 min to allow incorporation and activation of CM-H2DCFDA, followed by removing free dye and washing withprewarmed PBS. Phenol red-free DMEM/F-12 medium was added,and cells were again incubated at 37 °C for 60 min. Compounds wereadded as indicated, and fluorescence intensity (485/530 nm) wasmeasured with the POLARstar Omega (BMG Labtech) at 37 °C for22 h. Data were analyzed using GraphPad Prism 5.0.Mitochondrial Transmembrane Potential Assay. The mito-

chondrial transmembrane potential (ΔΨm) was investigated with theJC-1 assay (Cayman Chemicals, Biomol, Hamburg, Germany),according to the manual. This assay makes use of a lipophilic cationicdye (5,6, 5′,6′-tetrachloro-1,3,1′,3′-tetraethylbenzimidazolylcarbocya-nine iodide), which selectively enters into mitochondria and changesreversibly its color from red (JC-1 aggregates) to green (JC-1monomers) as the membrane potential decreases. Thus, HT-29 cellswere seeded in 96-well plates (TPP) at a density of 40 000 cells/well.One day later, cells were treated with the test compounds for 5 min at37 °C, followed by addition of the JC-1 dye for an additional 10 min.Cells were then washed twice with prewarmed JC-1 assay buffer toremove free JC-1 dye. Red (excitation 560/10 nm, emission 590/30

nm) and green (excitation 485/30 nm, emission 520/10 nm)fluorescence was measured in the POLARstar Omega (BMGLabtech). The ratio of red to green fluorescence was used as anindicator of mitochondrial transmembrane potential (ΔΨm). Datawere fitted using GraphPad Prism 5.0 according to the equation Y =Bottom + (Top − Bottom)/(1 + 10(LogIC50−X) × Hill slope), with a variableHill slope.

Mitochondrial Permeability Transition Pore Assay. Effects onthe MPTP were analyzed by use of the MitoProbe transition poreassay kit (Life Technologies), according to the manufacturer’sinstructions. Cells were loaded with a fluorescent calcein dye thataccumulates in the mitochondria. The fluorescence of cytosolic calceinwas quenched by addition of CoCl2, while mitochondrial fluorescencewas maintained. Opening of the MPTP leads to loss of mitochondrialcalcein fluorescence. For this purpose, trypsinized HT-29 cells weresuspended in HBSS/Ca buffer containing 10 nM calcein and 400 μMCoCl2 with a density of 10

6 cells/mL. Cells were pretreated with 2 μMcyclosporin A or vehicle control for 60 min at 37 °C and subsequentlytreated with the tested compounds for a further 30 min at 37 °C. Cellswere finally washed with HBSS/Ca buffer and analyzed by flowcytometry (Accuri C6, BD Biosciences), according to manufacturer’sinstructions. Analysis was performed using FlowJo 7.6 (Tree Star) andPrism 5.0 (GraphPad).

Oxygen Consumption Measurements. Oxygen consumptionwas determined by time-resolved fluorescence of an oxygen-sensitiveprobe (MitoXpress-Xtra HS, Luxcel Biosciences, Cork, Ireland). Theprobe fluorescence is quenched by molecular oxygen, so thatfluorescence lifetime increases with reduction in extracellular oxygenconcentration. One day before treatment, HT-29 cells were seeded in96-well plates (TPP) at a density of 80 000 cells/well. Medium wasremoved, and cells were incubated with 140 μL of prewarmed probediluted in phenol red-free DMEM/F-12 medium. Cells were incubatedat 37 °C for 10 min, and the test compounds were added. Finally, cellswere sealed with 100 μL of prewarmed HS mineral oil (LuxcelBiosciences) to prevent back diffusion of ambient oxygen. Time-resolved fluorescence was measured in the POLARstar Omega (BMGLabtech) apparatus with the following settings: temperature = 37 °C;TRF optic Z height = 6 mm; excitation = 380/20 nm; emission = 655/50 nm; window 1 (w1): 30 μs delay and 30 μs integration time;window 2 (w2): 70 μs delay and 30 μs integration time; interval time= 90 s; measurement time = 150 min. Background fluorescence wasmeasured in wells with medium and oil, but without cells and a probe.For data analysis, background fluorescence was subtracted for eachwell. Fluorescence lifetime (τ) was calculated by τ = 40/(ln(w1/w2))and plotted over treatment time. For comparing oxygen consumptionbetween treatments, the rate of probe fluorescence lifetime wasdetermined between 20 and 60 min and expressed relative tountreated cells. Data were analyzed using Prism 5.0 (GraphPad).

Extracellular Acidification Measurements. Extracellular acid-ification, which is mainly due to lactate production reflecting glycolyticactivity,26 was determined by time-resolved fluorescence of a pH-sensitive probe (pH Xtra, Luxcel Biosciences). Fluorescence lifetime ofthis probe increases with decrease in pH, so that it allowsmeasurement of extracellular acidification. One day before treatment,HT-29 cells were seeded in 96-well plates (TPP) with a density of80 000 cells/well and incubated at 37 °C overnight in a CO2-freeincubator. For subsequent measurements, the following low-bufferingaspiration medium was used according to the manufacturer’sinstructions: 1 mM PBS (pH 7.4), 20 mM glucose, 75 mM NaCl,54 mM KCl, 2.4 mM CaCl2, and 0.8 mM MgSO4. Before treatment,cells were washed twice with 200 μL of aspiration buffer and incubatedwith 140 μL of prewarmed probe diluted in aspiration medium. Cellswere incubated at 37 °C for 10 min, and the test compounds wereadded. Wells were not sealed with oil to avoid trapping of CO2reflecting Krebs cycle activity. Time-resolved fluorescence wasmeasured in the POLARstar Omega (BMG Labtech) apparatus withthe following settings: temperature = 37 °C; TRF optic Z height = 6mm; excitation = 380/20 nm; emission = 615/50 nm; window 1 (w1):100 μs delay and 30 μs integration time; window 2 (w2): 300 μs delayand 30 μs integration time; interval time = 100 s; measurement time =

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00072J. Nat. Prod. XXXX, XXX, XXX−XXX

J

200 min. Background fluorescence was measured in wells withmedium without cells. For data analysis, background fluorescence wassubtracted for each well. Fluorescence lifetime (τ) for each sample wascalculated by τ = 200/[ln(w1/w2)] and transformed to absolute pHvalues according to the equation pH = (1687.2 − τ)/199.12, asreported previously.26 The pH values were plotted over treatmenttime. For comparing different treatments, the extracellular acidificationrate was determined between 20 and 150 min. Data were analyzedusing Prism 5.0 (GraphPad).Statistical Analysis. Data are expressed as means ± standard error

of mean (SEM), if not otherwise noted. Statistical tests wereperformed using GraphPad Prism 5.0. For comparison of two groups,statistical significance was examined by unpaired two-tailed Student’s ttest. For multiple comparisons, data were analyzed by one-wayANOVA with subsequent Dunnett’s and Bonferroni post-test,respectively. A p value ≤ 0.05 was defined as statistically significantas given in the figure legends.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.jnat-prod.5b00072.

Additional information for the Experimental Section(PDF)GO network analysis for 2 (Figure S1) and the pKa

determination for 2 (Figure S2) (PDF)Global gene expression data (XLS)Global gene expression data (XLSX)1H NMR spectra for 2−7 (PDF)LC-MS spectra for 2−7 (PDF)

■ AUTHOR INFORMATIONCorresponding Author*Tel: +49 30 8413 1661. Fax: +49 30 8413 1960. E-mail:[email protected] Contributions∥C. Weidner and M. Rousseau contributed equally to thisstudy.NotesThe authors declare the following competing financialinterest(s): K. Siems and G. Hetterling are employees ofAnalytion Discovery, a company that sells natural products.

■ ACKNOWLEDGMENTSThe present work was supported by the German Ministry forEducation and Research (BMBF, grant numbers 0315082,01EA1303) and the European Union [FP7/2007-2011], undergrant agreement no. 262055 (ESGI).

■ REFERENCES(1) Anonymous. The Global Burden of Disease: 2004 Update; WorldHealth Organization: Geneva: 2008.(2) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman,D. Ca-Cancer J. Clin. 2011, 61, 69−90.(3) Shekhar, M. P. Curr. Cancer Drug Targets 2011, 11, 613−623.(4) Li, J. W.; Vederas, J. C. Science 2009, 325, 161−165.(5) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311−335.(6) Muller, M.; Kersten, S. Nat. Rev. Genet. 2003, 4, 315−322.(7) Weidner, C.; de Groot, J. C.; Prasad, A.; Freiwald, A.; Quedenau,C.; Kliem, M.; Witzke, A.; Kodelja, V.; Han, C. T.; Giegold, S.;Baumann, M.; Klebl, B.; Siems, K.; Muller-Kuhrt, L.; Schurmann, A.;Schuler, R.; Pfeiffer, A. F.; Schroeder, F. C.; Bussow, K.; Sauer, S. Proc.Natl. Acad. Sci. U. S. A. 2012, 109, 7257−7262.

(8) Weidner, C.; Wowro, S. J.; Freiwald, A.; Kawamoto, K.; Witzke,A.; Kliem, M.; Siems, K.; Muller-Kuhrt, L.; Schroeder, F. C.; Sauer, S.Diabetologia 2013, 56, 1802−1812.(9) Sauer, S. ChemBioChem 2014, 15, 1231−1238.(10) Mitscher, L. A.; Park, Y. H.; Alshamma, A.; Hudson, P. B.; Haas,T. Phytochemistry 1981, 20, 781−785.(11) Ghisalberti, E. L.; Jefferies, P. R.; Mcadam, D. Phytochemistry1981, 20, 1959−1961.(12) Meierhofer, D.; Weidner, C.; Hartmann, L.; Mayr, J. A.; Han, C.T.; Schroeder, F. C.; Sauer, S. Mol. Cell. Proteomics 2013, 12, 1965−1979.(13) de Groot, J. C.; Weidner, C.; Krausze, J.; Kawamoto, K.;Schroeder, F. C.; Sauer, S.; Bussow, K. J. Med. Chem. 2013, 56, 1535−1543.(14) Dat, N. T.; Lee, J.-H.; Lee, K.; Hong, Y.-S.; Kim, Y. H.; Lee, J. J.J. Nat. Prod. 2008, 71, 1696−1700.(15) Shi, H.; Ma, J.; Mi, C.; Li, J.; Wang, F.; Lee, J. J.; Jin, X. Int.Immunopharmacol. 2014, 21, 56−62.(16) Fuhr, L.; Rousseau, M.; Plauth, A.; Schroeder, F. C.; Sauer, S. J.Nat. Prod. 2015, 78, 1160−1164.(17) Kastan, M. B.; Bartek, J. Nature 2004, 432, 316−323.(18) Fadok, V. A.; Bratton, D. L.; Frasch, S. C.; Warner, M. L.;Henson, P. M. Cell Death Differ. 1998, 5, 551−562.(19) Elmore, S. Toxicol. Pathol. 2007, 35, 495−516.(20) Michalik, L.; Auwerx, J.; Berger, J. P.; Chatterjee, V. K.; Glass, C.K.; Gonzalez, F. J.; Grimaldi, P. A.; Kadowaki, T.; Lazar, M. A.;O’Rahilly, S.; Palmer, C. N.; Plutzky, J.; Reddy, J. K.; Spiegelman, B.M.; Staels, B.; Wahli, W. Pharmacol. Rev. 2006, 58, 726−741.(21) Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N. S.; Wang, J. T.;Ramage, D.; Amin, N.; Schwikowski, B.; Ideker, T. Genome Res. 2003,13, 2498−2504.(22) Nogueira, V.; Hay, N. Clin. Cancer Res. 2013, 19, 4309−4314.(23) Kroemer, G.; Galluzzi, L.; Brenner, C. Physiol. Rev. 2007, 87,99−163.(24) Chen, L. B. Annu. Rev. Cell Biol. 1988, 4, 155−81.(25) Fulda, S.; Galluzzi, L.; Kroemer, G. Nat. Rev. Drug Discovery2010, 9, 447−464.(26) Hynes, J.; O’Riordan, T. C.; Zhdanov, A. V.; Uray, G.; Will, Y.;Papkovsky, D. B. Anal. Biochem. 2009, 390, 21−28.(27) Cymes, G. D.; Ni, Y.; Grosman, C. Nature 2005, 438, 975−980.(28) Zhao, L.; Au, J. L.; Wientjes, M. G. Front. Biosci., Elite Ed. 2010,2, 241−249.(29) Bliss, C. I. Ann. Appl. Biol. 1939, 26, 585−615.(30) Vier, J.; Gerhard, M.; Wagner, H.; Hacker, G. Mol. Immunol.2004, 40, 661−670.(31) Gogvadze, V.; Orrenius, S.; Zhivotovsky, B. Trends Cell Biol.2008, 18, 165−173.(32) Loewe, S. Klin. Wochenschr. 1927, 6, 1077−1085.(33) Tallarida, R. J. J. Pharmacol. Exp. Ther. 2006, 319, 1−7.(34) Weidner, C.; Wowro, S. J.; Rousseau, M.; Freiwald, A.; Kodelja,V.; Abdel-Aziz, H.; Kelber, O.; Sauer, S. PLoS One 2013, 8, e80335.(35) Schmieder, R.; Edwards, R. Bioinformatics 2011, 27, 863−864.(36) Dobin, A.; Davis, C. A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.;Jha, S.; Batut, P.; Chaisson, M.; Gingeras, T. R. Bioinformatics 2013,29, 15−21.(37) Anders, S.; Pyl, P. T.; Huber, W. Bioinformatics 2015, 31, 166−169.(38) Robinson, M. D.; McCarthy, D. J.; Smyth, G. K. Bioinformatics2010, 26, 139−140.(39) Maere, S.; Heymans, K.; Kuiper, M. Bioinformatics 2005, 21,3448−3449.

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.5b00072J. Nat. Prod. XXXX, XXX, XXX−XXX

K