Studies of Jatrogossone A as a Reactive Oxygen ... - ACS Publications

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Studies of Jatrogossone A as a Reactive Oxygen Species Inducer in Cancer Cellular Models Taotao Ling,† Walter H. Lang,† Jane Craig,‡ Malia B. Potts,‡ Amit Budhraja,‡ Joseph Opferman,‡ John Bollinger,§ Julie Maier,† Travis D. Marsico,⊥ and Fatima Rivas*,† Department of Chemical Biology and Therapeutics, ‡Department of Cellular and Molecular Biology, and §Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105-3678, United States ⊥ Department of Biological Sciences, Arkansas State University, Jonesboro, Arkansas 72467, United States Downloaded via UNIV AUTONOMA DE COAHUILA on May 14, 2019 at 23:20:38 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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ABSTRACT: Natural products continue to provide a platform to study biological systems. A bioguided study of cancer cell models led us to a new member of the jatrophane natural products from Jatropha gossypiifolia, which was independently identified and characterized as jatrogossone A (1). Purification and structure elucidation was performed by column chromatography and high-performance liquid chromatography−mass spectrometry and NMR techniques, and the structure was confirmed via X-ray crystallography. The unique molecular scaffold of jatrogossone A prompted an evaluation of its mode of action. Cytotoxicity assays demonstrated that jatrogossone A displays selective antiproliferative activity against cancer cell models in the low micromolar range with a therapeutic window. Jatrogossone A (1) affects mitochondrial membrane potential (ΔΨm) in a time- and dose-dependent manner. This natural product induces radical oxygen species (ROS) selectively in cancer cellular models, with minimal ROS induction in noncancerous cells. Compound 1 induces ROS in the mitochondria, as determined by colocalization studies, and it induces mitophagy. It promotes also in vitro cell death by causing cell arrest at the G2/M stage, caspase (3/7) activation, and PARP-1 cleavage. The combined findings provide a potential mechanism by which 1 relies on upregulation of mitochondrial ROS to potentiate cytotoxic effects through intracellular signaling.

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systems featuring a trans-bicyclo[10.3.0]pentadecane framework, typically including hydroxylated or other oxygenated substituents. Such natural products have demonstrated a wide variety of biological properties including P-glycoprotein modulation7 and antiproliferative,8 antimalarial,9a anti-inflammatory,10 and antiviral properties.11 The main objective of the present work was to identify novel natural products as potential sources of anticancer agents, particularly for the treatment of drug-resistant cancers. Previously, the potential of natural products present in Jatropha isabellei Müll. Arg.9,10 (Euphorbiaceae) has been demonstrated particularly for jatrophone (2, Figure 1). Thus, a high-throughput screen of 100 terrestrial plant extracts including several from the genus Euphorbia was conducted. The secondary metabolites were extracted, fractionated, and

atural products historically have provided a direct source of molecular inspiration and a general platform for drug development.1 Their intricate molecular frameworks offer chemists uncharted chemotypes for the discovery of new molecular probes and potential drugs to advance understanding of the mechanisms of human disease.1−3 Natural products often feature biologically relevant pharmacophore patterns that have evolved as preferred ligand−protein binding interactions to cause an effect.4,5 The drug discovery program disclosed here focuses on natural products as sources of new chemical matter to study basic biological processes, which should lead to new druggable biological targets. The Euphorbiaceae family is a large group of flowering plants encompassing more than 7000 species.6 Several diterpenoid classes have been isolated from the genus Euphorbia (e.g., casbanes, jatrophanes, and lathyranes), displaying remarkable molecular complexity. The jatrophane and lathyrane diterpenoids are unique classes of macrocyclic © XXXX American Chemical Society and American Society of Pharmacognosy

Received: January 6, 2019

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DOI: 10.1021/acs.jnatprod.8b01087 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Although structurally similar in complexity to the core of jatrophone, compound 1 displays a less extended conjugated system than jatrophone, leading to the speculation that this compound would exhibit higher stability. In fact, in vitro absorption, distribution, metabolism, and elimination (ADME) studies comparing physicochemical properties of compounds 1 and 2 are consistent with such a model. Compound 1 shows improved stability with similar aqueous solubility to compound 2. Compounds 1 and 2 also show comparable binding affinity to human plasma proteins (86% and 74%, respectively) and display acceptable solubility, membrane permeability, and a reasonable efflux ratio in Caco-2 cells permeability assay9a (Table S1, Supporting Information). Compound 1 showed promising metabolic stability in human hepatic microsomes (t1/2 = 31 min), human plasma stability (t1/2 = 19.5 h), and simulated gastric stability (t1/2 = 124 h), while compound 2 has a short half-life in both human hepatic microsomes and human plasma (t1/2 = 0.10 and 0.13 h, respectively).9a The combined findings suggest organismal studies may be feasible for compound 1. Antiproliferation Properties of Compound 1. The lack of knowledge about the biological activity of compound 1 led to an investigation of its properties against cancer cellular models. The study focused on a subset of acute lymphoblastic leukemia (ALL) models, namely, KOPN-8 and SUP-B15, both of which carry specific genomic lesions. KOPN-8 carries the MLL-ENL fusion gene, and SUP-B15 was established from a pediatric patient with relapsed ALL (with the m-BCR ALL variant of the BCR-ABL1 fusion gene).18,19 The gene for the histone methyltransferase MLL participates in chromosomal translocations that eventually create MLL-fusion proteins associated with aggressive forms of childhood leukemia. The presence of some MLL rearrangements is an independent dismal prognostic factor, and patients diagnosed with them are typically categorized as being at high-risk.20 Therefore, identifying the natural product 1 as an antiproliferation agent will enable mechanistic studies to advance the drug discovery efforts in this area. A heat map of the antiproliferative activity following established protocols17 was generated (Table 1) using ALL stable cell lines representative of high-risk patient populations (KOPN-8, SEM, SUP-B15, UoC-B1), ALL murine models (BCR-ABL-WT and Bax/Bak DKO23), chronic myelogenous leukemia model (K562, BCR-ABL positive), aggressive non-Hodgkin lymphoma (Raji), and solid tumor models (HEPG2, MCF-7, MDA-MB-231, SUM149, U2OS), in comparison to the activity against normal cellular models (BJ, PMBC, and HMEC). The selectivity and potency of compound 1 toward specific cellular models could be attributed to the macrocyclic system with potential for 1,2 or 1,4 addition reactions at C-14 or C-12, respectively. Furthermore, the presence of the cyclopropyl group promotes potential for specific hydrophobic interactions and the potential for guanine/adenine adduct formation. Compound 1 was active in the low micromolar range (1−10 μM) against the human leukemic cells (KOPN-8, SEM, and UoC-B1) and murine cell models (BCR-ABL-WT, BCR-ABLDKO). Importantly, compound 1 was not cytotoxic in normal human cells at the tested concentrations (>100 μM), rendering it a good chemical tool with which to study cell death pathways in cancer cellular models. Compound 1 displayed moderate cytotoxicity activity (>10 μM) against solid tumor cell models (MDA-MB-231, SUM149, and U2OS). Findings were further

Figure 1. Selected natural products from the genus Jatropha.

evaluated for their biological properties against drug-sensitive and -resistant leukemia cells in a bioguided phenotypic screen, as previously described.9 Several fractions of the extract of Jatropha gossypiifolia L. (Euphorbiaceae) were identified as active hits. This species biosynthesizes diverse jatrophone-like structures and flavonoids with promising antibacterial and anticancer properties.10−12 Jatrophone (2, Figure 1), a macrocyclic molecule featuring several electrophilic centers was the most abundant compound isolated. Several research groups have demonstrated the potency of this compound against cancer cell models; however, its narrow therapeutic index limits its therapeutic value as a lead clinical candidate.9,10 Next, the related compounds, 9β-isabellione (3) and jatropholone A and B, were also identified, and they displayed modest biological properties.13b,14,16 This work focuses on jatrogossone A (1, Figure 1), which had not been reported at the time the present work commenced but was recently disclosed to be a minor component of J. gossypiifolia13b in a study that had not fully evaluated the compound’s biological properties in a broad range of cellular models. In this study compounds 1 and 2 were isolated in a 1:5 ratio. It is speculated that the American grown J. gossypiifolia favors the biosynthesis of 2 over 1. Moreover, this metabolite ratio varied based on the stages of plant growth (vegetative vs budding), with early stage producing primarily compound 2. Herein is a description of the biological evaluation of the diterpenoid jatrogossone A (1, Figure 1) isolated from J. gossypiifolia cultivated in Tennessee, United States, through a bioguided fractionation protocol against cancer cellular models. The findings include a thorough evaluation of compound 1 as a selective radical oxygen species (ROS) inducing agent against cancer cellular models with minimal effects to normal cells. This ROS event can be prevented by using N-acetyl cysteine (NAC), suggesting that compound 1 interacts in a reversible manner with its cellular target (s).

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RESULTS AND DISCUSSION A crude J. gossypiifolia was prepared from a Soxhlet extraction of the dried plant material (50 g) with ethanol, and then bioguided fractionation was conducted to assess antiproliferative activity in several cancer cell models via a cell proliferation assay (CellTiter Glo as previously established).16 The active fractions were purified by column chromatography prior to reversed-phase high-performance liquid chromatography (RP-HPLC) purification. Along with compound 1, other reported constituents including jatrophone, jatropholones, and flavones were detected by mass spectra and by comparison of their NMR spectra with those in the reported literature.14−16 The identification of compound 1 was conducted using twodimensional (2D) NMR and was unequivocally confirmed by X-ray analysis,13a which was in full agreement with the recently disclosed report by Zhang.13b B



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Table 1. Bioactivity Heat Map of Compounds 1, 2, and 3 using Established Proliferation Protocols17 a

Figure 2. Representative KOPN-8 cell cycle analysis. Cells were treated with vehicle control (DMSO), positive control (2 μM staurosporine), or compound 1 (10 μM) for 24 h. Representative ModFit plots are found in the Supporting Information). All graphs represent mean and standard deviation (SD) of at least three experiments. ****p < 0.0001, ***p < 0.001 according to two-way ANOVA.

compound 1 treatment, confirming an expectation of apoptosis induction in a time-dependent manner. To complement Annexin V staining, caspase activity was monitored to evaluate whether the mechanism of cell death involved cleavage of caspase 3/7. Increased caspase 3/7 activity corresponding with decreased viability of both KOPN8 and U2OS cells occurred in a dose-dependent manner (Figure 4), substantiating the cell death-inducing effects of compound 1. Similar effects have been reported for compound 2 in other cellular models.9b Furthermore, cotreatment studies of compounds 1 and 2 with the pan-caspase inhibitor Z-VADFMK or the necrosis inhibitor necrostatin-1 in KOPN-8 cells were conducted (Figure S3, Supporting Information). The presence of Z-VAD-FMK significantly diminished cell death for compounds 1 and 2, suggesting that apoptosis is dependent on caspase activity and that both compounds might undergo similar cell death mechanisms. Then, the level of apoptosis-related proteins in response to treatment with compounds 1 and 2 was analyzed by Western blotting (WB) in both KOPN-8 and MCF-7 cancer cells. As shown in Figure 5, caspase-7 and PARP-1 cleavage were both observed in a time and concentration-dependent manner. Menadione (Men) was used as a positive control at the concentration of 10 μM in KOPN-8 and at 30 μM in MCF-7. These results indicated that compounds 1 and 2 may cause cell death via the apoptosis-related proteins PARP-1 and caspase3/7 in the cancer cell models used. Cleavage of PARP-1 was observed after 24 h of treatment with compound 1 in both cell lines tested. However, for compound 2, PARP-1 cleavage was detected after 3 h of treatment. A dose-dependent increase in levels of cleaved PARP-1 corresponded to decreased levels of full-length caspase 3/7 supporting the notion that apotosis is the likely mechanism of cell death upon treatment with compounds 1 and 2. The BCL-2 family of proteins is a gatekeeper to the apoptotic response. These proteins act as both anti-apoptotic and pro-apoptotic regulators. To assess the anti-apoptotic response, the effect of compound 1 was further studied in leukemic cell lines overexpressing anti-apoptotic proteins (MCL-1, BCL-2, BCL-XL, and BCL-W) and triple knockout cell line (TKO-hMCL-1) generated as previously described by

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ND: not determined.

validated by a propidium iodide (PI) assay for breast cancer cell models (Figure S2, Supporting Information). Compound 2 showed partially potent activity across all the tested cellular models (hematological and solid tumor cells), but it had a narrow therapeutic index (cytotoxicity against PMBC/HMEC cells) in agreement with previous biological studies.21 Compound 3 (9β-Isabellione16) also showed activity against BCR-ABL-WT and KOPN-8 cell lines. To determine whether the inhibition of cell viability by compound 1 was due to effects on cell-cycle progression, the compound’s effects in KOPN-8 were investigated (Figure 2). Incubation of compound 1 with KOPN-8 resulted in a significantly increased percentage of cells (∼10%) in the G2/ M phase and a reduced percentage of cells (∼5%) in G1/G0 and S phase. Similar results have been reported in nonsmall cell lung cancer cells treated with the natural product xanthatin.22 Progression of cells from the G2 to M phase is dependent upon an intact genome, and failure to overcome G2/M arrest results in apoptosis. The compound staurosporine was used as a positive control, which showed cell arrest in the S phase in this cellular model. Next, it was determined that compound 1 induces programmed cell death. KOPN-8 cells were double-stained with Annexin V and PI dyes to determine the percentages of cells in early and late apoptosis, and the percentage of viable cells. As shown in Figure 3, the percentage of treated cells in early apoptosis (Annexin+, PI-) was not appreciably different from that of dimethyl sulfoxide (DMSO)-treated cells, but the percentage of treated cells in late apoptosis significantly increased (∼15%), and that of live cells decreased, after 24 h of C



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Figure 3. Measurement of cell death by Annexin V−FITC binding and PI uptake. Representative KOPN-8 plot of three independent biological replicates after 24 h. (A) Vehicle control (DMSO). (B) Positive control (2 μM staurosporine). (C) Compound 1 (10 μM).

Figure 4. ApoTox-Glo Triplex assay in cancer cell models to determine viable cells (GF-AFC), apoptotic cells (caspase 3/7), or cytotoxicity by examining membrane integrity (bis-AAF-RF110) after treatment with compound 1 (100−0.15 μM). (A) KOPN-8 cells. (B) U2OS cells. Graphs show that compound 1 decreases cellular viability while increasing apoptotic activity. Bars depict mean ± standard error of measure (SEM) of three independent experiments.

Figure 5. Evaluation of apoptosis-related proteins by treatment with compounds 1 and 2. (A) KOPN-8. (B) MCF-7 cell models. The blots show a representative of three independent experiments.

thylrhodamine methyl ester (TMRM+), using flow cytometry to determine the effects of compounds 1 and 2 on mitochondrial membrane integrity.24 TMRM+ is a noninvasive cationic fluorescent dye used to quantify changes in mitochondrial membrane potential. It accumulates in the negatively charged mitochondria by electrostatic attraction.

Kross et al.23 Compound 1 was selectively cytotoxic toward MCL-1 overexpressing cells (Figure 6) with an IC50 value of 3.3 μM, while the triple knockout model (TKO-hMCL-1) displayed an IC50 of 19.5 μM. Next, the mitochondrial membrane potential (ΔΨm) was evaluated by probing the fluorescence intensity of tetrameD



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recorded after 48 h (Figure 7D). Compound 2 showed a shift in mitochondrial membrane potential at 4 μM with substantial cell death after 24 h (data not shown). Interestingly, for compound 1, a small shift in mitochondrial membrane potential was measured at 1−3 μM with no change at 10 μM (see Supporting Information). Such results can be attributed to the fact that substantial cell death was recorded during treatment. Neither compound caused statistically significant changes in the MCF-7 cellular model (see Supporting Information). Thus, compounds 1 and 2 display mitochondria depolarization properties in a cell-type and timedependent manner. Compound 1 Induces ROS in Live-Cell Models. Eukaryotic cells have highly organized pathways to orchestrate the many extracellular stimuli received and their conversion into specific physiological processes. This classical cascade, termed signal transduction pathways, includes a series of events occurring constitutively and initiated by interaction of a ligand with its receptor on the cell membrane. ROS have been reported in both solid and hematopoietic cancers, where they are associated with tumor development and progression.25,26 Cancer cells also express antioxidant proteins to detoxify ROS, suggesting that fine-tuning intracellular ROS signaling is critical for cancer cell survival. ROS levels are regulated by redox enzymes and reducing factors (i.e., glutathione). Excess levels of ROS can lead to DNA and cellular damage. Cancer cells have a higher metabolic activity and ROS levels than noncancerous cells. ROS contribute to cancer cell homeostasis and growth. However, treating cancer cells with ROS-inducing anticancer agents exceeds the threshold for ROS, resulting in activation of multiple cell-death pathways, including the

Figure 6. Evaluation of compound 1 against BCR-ABL (murine and human) leukemic cells. The graph depicts the means and SD of at least two independent experiments.

Thus, a shift in fluorescent intensity corresponds to a depolarization event due to a change in the dye’s concentration in the mitochondria. 4′,6′-Diamidino-2-phenylindole (DAPI) was used to categorize any compromised cells. Representative data from KOPN-8 are shown in Figure 7, including DMSOtreated negative control cells (Figure 7A) and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)-treated positive control cells (Figure 7B). Compound 1-treated cells (Figure 7C) showed a small shift in membrane potential at 30 μM after 24 h. However, significant depolarization was

Figure 7. TMRM assay representative of three biological replicates evaluation of compound 1 in KOPN-8 cells. (A) DMSO. (B) FCCP (10 μM) treatment for 0.3 h. (C) Compound 1 (30 μM) for 24 h. (D) Compound 1 (30 μM) for 48 h. E



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Figure 8. ROS formation in MCF-7 and U2OS cells. (A) MCF-7 breast cancer cells or (B) U2OS osteosarcoma cells were treated with compound 1 (30 μM), compound 2 (30 μM), or menadione (Men, 10 μM) as a positive control. NAC (100 μM) was added to inhibit ROS formation. All graphs represent mean and SD of at least three experiments. ****p < 0.0001, ***p < 0.001 according to two-way ANOVA.

inhibition of mammalian target of rapamycin (mTOR) signaling and downregulation of specific proteins.26 Therefore, understanding the susceptibility of cancer cells to oxidative signals could open a new therapeutic opportunity for rational design of novel anticancer agents. ROS in this cascade have been proposed as second messengers in the activation of signaling events that lead to cellular survival or death.27 Moreover, redox-sensitive cysteine residues are known to sense and transduce changes in cellular redox status caused by ROS production and oxidized thiols. Previous reports indicate that compound 2 is prone to nucleophilic addition reactions with reactive cysteines.9a A high level of oxidative phosphorylation is a liability to tumor cells because of mitochondria’s role in apoptosis and because of ROS generation. If the compounds elevate ROS, they can trigger intrinsic apoptotic pathways. To further examine the effects of compound 1 and gain understanding on how it causes cell death, ROS evaluation experiments were conducted to compare compound 1 to menadione (Men) and compound 2. In control wells, the precursor NAC was added to prevent ROS formation. ROS levels in each well were evaluated by using CellROX green reagent. This cell-permeable dye is weakly fluorescent in a reduced state and exhibits bright green photostable fluorescence upon ROS oxidation (with absorption/emission maxima of 485/520 nm).28 Significant ROS induction was observed in MCF-7 and U2OS cells by compound 1 treatment (Figure 8). Compound 1 induces ROS formation in both cell lines at levels comparable to the positive control (menadione), whereas compound 2 is less effective. NAC partially quenched ROS formation in both cell lines. The ROS production by compound 1 was much lower in human mammary epithelial cell (HMEC) normal cells, identifying this compound as a good chemical probe with which to study ROS effects in cancer cells (Figure 9). Cell detachment occurred upon treatment with compound 2 (data not shown). For live-cell imaging, the MDA-MB-231 cell model was treated with compound 1 (Figure 10A) or DMSO (Figure 10B) to visualize the localization of ROS levels in the cells.29 The cells were also stained with the organelle MitoTracker Deep Red. Treatment with compound 1 resulted in ROS colocalizing in the mitochondria (white, merge). The cells exposed to NAC are distinctly protected against ROS, as it presumably increases glutathione levels, which bind to either

Figure 9. ROS formation in HMECs. Primary cells treated with Men (10 μM, positive control) or compound 1 (30 μM), with or without NAC. Bars depict mean ± SEM three independent experiments. ****p < 0.0001, ***p < 0.001 according to Student’s t test.

compound 1 or the toxic breakdown products generated by the compound. To further assess the latter possibility, liquid chromatography−mass spectrometry (LC-MS) and NMR analyses of compound 1 in CH3CN or DMSO with NAC or glutathione were conducted. Incubation for 16 h at 37 °C revealed that compound 1 did not yield any Michael addition adduct, and intact compound 1 was recovered. Mitochondria serve a vital role in the cell as a center for energy generation in the form of adenosine triphosphate (ATP) via oxidative phosphorylation and organizing metabolic cascades that drive many cellular functions. Uncontrolled ROS can lead to loss of membrane potential, leading to damaged mitochondria. Mitochondria undergoing prolonged or irreparable damage are eliminated via mitophagy, a form of selective autophagy.30 Because of the observed induction of ROS, compound 1 was tested for its ability to induce mitophagy events (Figure 11). Mitophagy was measured by using a transfected mKeima-RedMito-7 reporter, which is the fluorescent Keima protein tagged with a mitochondrion targeting signal peptide sequence in U2OS cancer cell model.31 Keima is a unique fluorescent protein that responds to changes in pH value: at neutral pH, it is activated at 440 nm to yield pseudocolored pink, but under acidic conditions, Keima is activated at 560 nm to yield pseudocolored green, distinguishing between cytoplasmic mitochondria (shown in F



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Figure 10. Live-cell ROS colocalization studies. (A) MDA-MB-231 cells treated with compound 1 (30 μM). (B) Cells treated with DMSO for 1 h, then stained with CellROX green reagent, MitoTracker Deep Red, and nuclear stain Hoechst 33342 (blue). Representative images of two independent experiments imaged at 63× magnification.

Figure 11. Transfected mKeima U2OS cell model imaged at 60× magnification. (A) DMSO. (B) Compound 1 (1.25 μM). (C) compound 1 (2.5 μM). (D) Compound 2 (1.25 μM). (E) Compound 2 (2.5 μM). (F) Relative quantification. Bars depict the mean and SD of at least three independent experiments. ***p < 0.0006 and *p < 0.032 according to Student’s t test.

H2O2 into its inert components, plays an important role in determining the cancer-killing ability of certain natural products. For example, Klingelhoeffer et al. reported that, when H2O2 is applied to different human cancer cell lines, cell viability decreases as the concentration of catalase decreases.33 Hence, catalase, SOD1, and Trx expression levels were evaluated upon treatment with compound 1 to determine the response of the oxidative stress defense system upon ROS induction. Western blots (WB) were used to measure any potential changes of Trx levels in KOPN-8 and MCF-7 upon treatment with compounds 1 or 2. Unlike Trx, neither catalase nor SOD1 could be detected reliably by WB, presumably due to low expression levels in those cell lines. In the presence of compound 1, there was a clear time and dose-dependent increase of Trx levels at the 3 and 6 h time point (Figure 12).

pink) and mitochondria in the last stages of mitophagic degradation (shown in green). Treating U2OS cells with either compound 1 or 2 at 1.25 μM or 2.5 μM for 8 h induced mitophagy. However, compound 1 led to more mitophagy induction at 2.5 μM, as shown in the relative quantification (Figure 11F). Thus, these data strongly suggest that compound 1 can induce mitophagy that correlates with ROS induction in cancer cell models. Importantly, the mitophagy-inducing concentration is at least fivefold lower than the concentration required to induce apoptosis in U2OS. Hence, compound 1 is a promising chemical probe with which to study mitophagy in cancer. To defend against foreign entities or natural products, cells modulate the activity of redox-related enzymes [catalase, superoxide dismutase 1 (SOD1), and thioredoxin (Trx)].32 Indeed, activity of the enzyme catalase, which breaks down G



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Figure 12. Evaluation of oxidative stress defense system. (A) WB of MCF-7 cells treated with compounds 1 (10−100 μM) and 2 (1−10 μM) for 3 and 6 h, respectively, followed by probing with tubulin and thioredoxin specific antibodies. (B) Quantification of thioredoxin signals normalized to tubulin levels in each sample. Bars depict mean and SD of at least three independent experiments. Jatrophone (2). White solid; [α]25D +293 (c 0.01, EtOH); 1H and C NMR as previously reported;9 HRESI-TOF-MS (positive) m/z 313. 1805 [M+ H]+ (calcd for C20H24O3, 313.1803). Cytotoxicity Assay. All cell lines were incubated at 37 °C and were maintained in an atmosphere containing 5% CO2 using proper sterile cell culture practices.35 Cell lines were purchased from American Type Culture Collection (ATCC) or Leibniz-Institute Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ). Adherent cells (MDA-MB-231, SUM149, MCF-7, U2OS, HEPG2, and normal cell BJ, CRL-2522) were grown to 80%−90% confluence, and suspension cells were grown to the densities recommended by ATCC/DSMZ before use. Cells were cultured in RPMI (ATCC 30-2001), DMEM, (ATCC, 30-2002), Ham’s 12 (ThermoFisher 11765-054), EMEM (ATCC 30-2003), or McCoy’s 5a (ATCC 30-2007) media supplemented with 10% fetal bovine serum (FBS, Hyclone). Primary mammary epithelial normal (HMEC, ATCC PCC-600-010) were grown in 96-well plates in cell basal medium (ATCC PCS-600-030) and growth kit (ATCC PCS-600040) according to ATCC. The human leukemia cell lines, KOPN-8, SEM, SUP-B15, PBMC, UoC-B1, and murine BCR-ABL (WT: wild type, DKO: Bax and Bak double knockout) cell models23 were cultured as described.29 CellTiterGlo (CTG) Luminescent Cell Viability Assay kit (G7570, Promega) was performed as described before.9,17 The positive controls included staurosporine (25 μM), gambogic acid (10 μM), and a toxic quinoline, namely, N1-(5-fluoro2-methoxybenzyl)-N3-(7-(3-(trifluoromethyl)benzyl)quinolin-4-yl)propane-1,3-diamine. Annexin V-FITC Apoptosis and Cell Cycle. The murine model BCR-ABL leukemic B cell overexpressing anti-apoptotic proteins (MCL-1, BCL-2, BCL-XL, and BCL-W) and the human TKOhMCL-1 were evaluated as described.23 The samples were probed with AnnexinV−fluorescein isothiocyanate (FITC) (Roche/Boehringer Mannheim) according to the manufacturer’s instructions. Briefly, KOPN-8 or SUB-P15 cells were plated (1.00 × 106 cells/ plate) and incubated for 12 h at 37 °C. Cells were treated with compound 1 or controls for 24 h, then stained with AnnexinV-FITC, PI, or DAPI. Cells were fixed with cold methanol, treated with RNase, and stained with PI solution (50 μg/mL). The Annexin profiles and cell-cycle distribution were determined with FACSCalibur analyzer (BD Biosciences) and analyzed with Cell-Quest software and ModFitsoftware (Verity Software House). Propidium Iodide Assay. The assay was conducted with MDAMB-231 and Sum149 cancer cell models seeded at 6 × 104 to 2 × 105 cells/well (CLS3548, Corning) and cultured for 24 h, then treated with compound 1 or 2 or 48 h. Cells were fixed with cold methanol and stained (0.4% PI, Sigma-Aldrich). Fluorescence was measured and anlyzed with Lionheart FX (BioTek). Cell viability was calculated/plotted as percent of surviving cells after treatment relative to vehicle wells (GraphPad Prism 7.0). ApoTox-Glo Triplex Assay. Experiments were conducted according to the manufacturer’s instructions (G6320, Promega). The cells were incubated for 12 h at 37 °C, then treated with compounds for 24 h. Viability/Cytotoxicity reagent was added and

For compound 2, a similar increase of Trx expression was detected at the lowest concentration. However, the Trx levels decreased at high doses of compound 2 treatment, presumably due to the induction of cell death at those concentrations, and Trx expression was depleted at 24 h (data not shown). A similar expression pattern was observed for compound 2treated KOPN-8 cells (Figure S9, Supporting Information).

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EXPERIMENTAL SECTION

General Experimental Procedures. A P-1020 polarimeter (JASCO) was used to measure optical rotations (2 mL cell with a 100 mm path length). A Waters Xevo G2 QTOF mass spectrometer was used to record high-resolution electrospray ionizatin time-of-flight mass spectrometry (HRESI-TOF-MS) data. NMR spectra were recorded on Bruker AVANCE III 500 spectrometer. Preparative HPLC separations were performed on Waters PrepLC system with a Symmetry C18 column (19 × 300 mm, 7 μm) with Shimadzu LC-8A binary preparative pump and a Shimadzu SCL-10A VP system controller connected to a Gilson 215 auto sampler and Gilson 215 fraction collector (Gilson, Inc.). Detections were performed by a Shimadzu SPD-M20A photodiode array detector and a Shimadzu ELSD-LT II evaporative light scattering detector (Shimadzu Corp.). Silica gel (60, particle size 0.040−0.063 mm, Merck) or Biotage Isolera Four with normal-phase silica gel was employed for column chromatography. Precoated silica gel 60 F254 plates were used for thinlayer chromatography (TLC) with detection under 254 and 365 nm. Plant Material. Plants of Jatropha gossypiifolia grown in Memphis, TN, United States, were collected in September 2016. The plant was identified34 by Dr. Travis D. Marsico at Arkansas State University, and a voucher specimen was deposited at the Arkansas State University Herbarium (STAR), with the acession number STAR034649. Extraction and Isolation. Whole plant material (50 g) was dried, minced, and underwent Soxhlet extraction in refluxing ethanol (3 × 250 mL) for 48 h. Fractionation of terrestrial plants was performed via a method previously established.17,29 The mixture was filtered, and the solvent was evaporated to afford the crude mixture as brown syrup, which was purified via silica gel column chromatography eluted in a gradient of increasing polarity (hexanes−EtOAc, 100:1, 80:1, 50:1, 30:1, 2:1, 0:1, v/v), and fractions (1−30) were separated and evaluated for biological activity. The nonpolar fractions (1−14) obtained were pooled and subjected to normal-phase silica gel column chromatography (hexanes−EtOAc, 95%−85%) and further purified via semipreparative HPLC. HPLC sequence, methanol in H2O (with 0.1% trifluoroacetic acid (TFA)): 0 min, 98:2; 0.5 min, 98:2; 6.5 min, 0:100; 12.3 min, 0:100; 12.5 min, 98:2; 12.95 min, stop. The flow rate was 25 mL/min. The major components isolated from the active fractions were jatrophone (compound 2, 15 mg, 0.03% by mass) and jatrogossone A (compound 1, 3 mg, 0.006% by mass). Jatrogossone A (1). Yellow oil; [α]25D +50.39 (c 3.60, CHCl3); 1H and 13C NMR as previously reported;13b HRESI-TOF-MS (positive) m/z 381. 2039 [M+ Na]+ (calcd for C22H30NaO4, 381.2042). H



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briefly mixed by orbital shaking (300−500 rpm for ∼30 s), and plates were incubated for 30 min at 37 °C. Fluorescence was measured at 400Ex/505Em (viability) and at 485Ex/520Em (cytotoxicity). Then, Caspase-Glo 3/7 reagent was added, and plates were mixed by orbital shaking (300−500 rpm for ∼30 s), followed by 0.5 h of incubation at room temperature, and luminescence was recorded in an Envision plate reader (PerkinElmer). Tetramethylrhodamine Methyl Ester Perchlorate (TMRM) Assay. Image-iT TMRM Reagent (Invitrogen, No. I34361) was used following manufacturer’s instruction. KOPN-8 cells (4 × 104 or 5 × 105 cells/well) or MCF-7 cells (2 × 104 or 2 × 105 cells/well) were seeded in six-well plates for 24 h, then treated with compound 1, 2, or vehicle for 24−48 h. FCCP (10 μM) was used as the positive control and was added for 10 min prior to TMRM staining (100 nM for 0.5 h). Medium was removed, and cells were washed with phosphatebuffered saline (PBS), trypsinized, or resuspended in PBS supplemented with 1% FBS and analyzed by flow cytometry. ROS Measurement. MCF-7 (wild type or stable transfected mRuby-Mito-7),29 MDA-MB-231, and U2OS cell lines were evaluated with CellROX green reagent (Molecular Probes No. C10444) according to manufacturer’s protocol. Cells were treated with compounds of interest or controls [DMSO 0.5% (v/v) as a negative control, Men (10−30 μM) as a positive control, and with or without N-acetyl cysteine (50−500 μM) as a free radical scavenger] for 1 h at 37 °C. Green reagent was added to a final concentration of 5 μM, and cells were incubated for an additional 30 min. Cells were washed with PBS, fixed with 4% (v/v) paraformaldehyde for 15 min, and washed with PBS. Fluorescence intensity was measured in a Clariostar plate reader (BMG LABTECH) at 535 nm or an Envision plate reader (PerkinElmer using FITC settings Exc FITC 485 and Ems FITC 535). Live Cell Confocal Microscopy. ROS assays were conducted in eight-well chamber (ibidi μ-slides No. 80826). Cells were plated at 4 × 104 per well in phenol red free medium and incubated at 37 °C for 12 h. For rescue studies, cells were pretreated with NAC (100 μM) for 0.5 h before adding compound 1 (10−30 μM) or Men (10−30 μM as a positive control) for 1 h, followed by addition of CellROX green reagent (Molecular Probes No. C10444) to a final concentration of 5 μM. For colocalization studies, MitoTracker Deep Red FM (M22426, Invitrogen) was added at 250 nM. Nuclear stain Hoechst33342 (H3570, Invitrogen) was added to a final concentration of 500 nM, 0.5 h prior to evaluation of the cells. Then, cells were washed twice with phenol red free medium, followed by live imaging on a 3i Marianas Spinning Disk confocal microscope system at a magnification of 63× and a resolution of 512 × 512 pixels. Mitophagy Assay. The mKeima-Red-Mito-7 plasmid was obtained from Addgene (No. 56018 from Michael Davidson). A stable clone of U2OS was generated by transfecting cells (1.5 × 105 ATCC) with plasmid (2.0 μg) containing a neomycin selection construct using Effectene Transfection Reagent (Qiagen) following the manufacturer’s protocol. Transfected cells were selected using G418 (250 μg/mL) for two weeks, sorted by flow cytometry and maintained under G418 pressure thereafter. U2OS cells expressing mKeima-Red-Mito-7 were plated at a density of 40 000 cells per well in Nunc Lab-Tek II 4 well chamber slides and incubated overnight at 37 °C. Cells were treated with compounds 1 and 2 for 8 h at 37 °C. DMSO (0.5%) was used as a negative control. Live cells were imaged on a Leica SP8 TCS (TCS = true confocal scanning, at 60× magnification). Mitophagy levels were quantified using a ratio of fluorescence intensity of mitophagy events to fluorescence intensity of cytoplasmic mitochondria. Western Blot Analysis. Cell lysates were prepared with lysis buffer [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycolbis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1% (v/ v) Triton X-100, 0.5% w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, and 10% (v/v) glycerol] supplemented with protease inhibitor cocktail (cOmplete, and PhosSTOP, Roche). Lysates were placed on ice for 5 min, then centrifuged at 17 000 rpm at 4 °C for 10 min. Total protein concentration was measured with

Pierce BCA protein assay (ThermoFisher Scientific). Total protein lysate (30 μg per lane) was separated by gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) on 4%−12% BisTris-NuPage polyacrylamide gels using 1X MES as running buffer (ThermoFisher Scientific) and transferred to nitrocellulose membranes (0.22 μM pore size, LI-COR Biosciences). After nonspecific binding was blocked with 5% nonfat dry milk, membranes were probed with the following monoclonal or polyclonal primary antibodies according to the manufacturer’s recommendations: antiPARP-1 (Cell Signaling No. 9542), anti-β-tubulin (Neo Markers No. MS1226PABX), anti-Caspase 3 (Cell Signaling No. 9662), antiCaspase 7 (Cell Signaling No. 9494), Oxidative Stress Defense (Catalase, SOD1, TRX, smooth muscle Actin), and Cocktail (Abcam No. ab179843). For detection, membranes were incubated with secondary antibodies (goat antirabbit-IgG labeled with IR-Dye 680RD or goat antimouse-IgG labeled with IR-Dye800CW [(LICOR Biosciences), at a dilution of 1:15 000 each] and scanned with a LI-COR Odyssey system (LI-COR Biosciences). Statistical Analysis. At least two independent biological replicates were conducted in three technical replicates for each experimental condition. The mean luminescence of each experimental treatment group was normalized as a percentage of the mean intensity of untreated controls. EC50 values (μM) were calculated by Pipeline Pilot Software (Accelrys). For cell viability, cell-cycle progression, ROS, and Western blot assays, the analyses were performed via oneway or two-way ANOVA with post-testing for each condition (concentration, cell cycle stage, apoptotic event, cell line) or according to Student’s t test using GraphPad Prism 7.0 (GraphPad Prism Software Inc.).

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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.8b01087. ADME table, representative cytotoxicity graphs, 1H NMR and 13C NMR spectra of compound 1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 901-595-6504. E-mail: [email protected]. ORCID

Fatima Rivas: 0000-0003-3643-2035 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by ALSAC St. Jude Children’s Research Hospital. The authors are thankful for the Analytical Technologies Center Core Facility, the Flow and Cell Cycle Facility, and the Cell and Tissue Imaging Center of St. Jude Children’s Research Hospital. All centers were supported in part by the Cancer Center Support Grant (P30CA021765) from the U.S.A. National Cancer Institute, NIH, Bethesda, MD.

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REFERENCES

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