Article pubs.acs.org/jnp
Sampangine (a Copyrine Alkaloid) Exerts Biological Activities through Cellular Redox Cycling of Its Quinone and Semiquinone Intermediates Fakhri Mahdi,† J. Brian Morgan,† Wenlong Liu,†,‡ Ameeta K. Agarwal,§,⊥ Mika B. Jekabsons,∥ Yang Liu,† Yu-Dong Zhou,*,†,∇ and Dale G. Nagle*,†,‡,⊥ †
Department of Biomolecular Sciences, §National Center for Natural Products Research, and ⊥Research Institute of Pharmaceutical Sciences, School of Pharmacy, ∥Department of Biology, and ∇Department of Chemistry and Biochemistry, University of Mississippi, University, Mississippi 38677, United States ‡ School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan Province 410208, People’s Republic of China ABSTRACT: The cananga tree alkaloid sampangine (1) has been extensively investigated for its antimicrobial and antitumor potential. Mechanistic studies have linked its biological activities to the reduction of cellular oxygen, the induction of reactive oxygen species (ROS), and alterations in heme biosynthesis. Based on the yeast gene deletion library screening results that indicated mitochondrial gene deletions enhanced the sensitivity to 1, the effects of 1 on cellular respiration were examined. Sampangine increased oxygen consumption rates in both yeast and human tumor cells. Mechanistic investigation indicated that 1 may have a modest uncoupling effect, but predominately acts by increasing oxygen consumption independent of mitochondrial complex IV. Sampangine thus appears to undergo redox cycling that may involve respiratory chain-dependent reduction to a semi-iminoquinone followed by oxidation and consequent superoxide production. Relatively high concentrations of 1 showed significant neurotoxicity in studies conducted with rat cerebellar granule neurons, indicating that sampangine use may be associated with potential neurotoxicity.
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affect mitochondrial function, especially that of the ATP synthase.11 To test the hypothesis that 1 disrupts mitochondrial function, cell-based respiration studies were performed to determine the effects of 1 on cellular respiration. In S. cerevisiae and human breast tumor T47D cells, 1 increased the rate of oxygen consumption. Inhibitor and substrate-based mechanistic studies indicated that 1 primarily increases non-mitochondrial oxygen consumption, promotes mitochondrial redox cycling, and may partially act as an atypical protonophore-type uncoupler. This study suggests that the biological activities attributed to 1 may arise from a quinone-like redox cycling mechanism that consumes oxygen and produces high levels of cellular ROS.
he copyrine alkaloid sampangine (1) has gained considerable interest for its antifungal/agrochemical,1−3 antibacterial,4 antiparasitic,1 and potential antitumor5−7 activities. First isolated from an extract of the stem bark of the cananga tree (or “yling−yling”), Cananga odorata Hook, F. and Thoms. (Annonaceae),8 1 was shown to have potent antifungal activity.9 Numerous studies have focused on elucidating the mechanism(s) of action for antifungal activity. Early studies indicated that the bactericidal, fungicidal, and cytotoxic activities of 1 may be related to reactive oxygen species (ROS)-mediated mechanisms.5,6 In human leukemic HL-60 cells, 1 induced an apoptotic response that appeared to involve a ROS-associated biphasic, concentration-dependent, alteration in mitochondrial membrane potential (ΔΨm).5,6 Transcriptional profiling coupled with mutant analysis studies indicates that 1 triggers a genomic response indicative of hypoxia in the budding yeast Saccharomyces cerevisiae.10 Mutants with a defective heme biosynthetic pathway exhibited increased 1 sensitivity, and exogenous hemin supplementation partially rescued wild-type cells from the growth inhibition imposed by 1.10 A recent functional genomic-based study revealed that 1 inhibits heme biosynthesis, most likely via the hyperactivation of uroporphyrinogen III synthase in the heme biosynthetic pathway.11 Genome-wide screening of yeast deletion mutant libraries revealed that 1 hypersensitivity is associated with mutants that © XXXX American Chemical Society and American Society of Pharmacognosy
Received: September 11, 2015
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DOI: 10.1021/acs.jnatprod.5b00819 J. Nat. Prod. XXXX, XXX, XXX−XXX
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RESULTS AND DISCUSSION
respiration was examined in T47D cells (Figure 1C). An inhibitor of F0F1-ATPase, oligomycin decelerates mitochondrial electron transport (reflected by a decrease in the rate of oxygen consumption) by increasing the proton gradient across the inner mitochondrial membrane. The uncoupler FCCP [2-([4(trifluoromethoxy)phenyl]hydrazinylidene)propanedinitrile] stimulates state 4 respiration (Figure 1D) by dissipating the proton gradient across the mitochondrial inner membrane. Compound 1 overcame oligomycin-stalled cellular oxygen consumption in both T47D (Figure 1C) and the human hepatoma Hep3B cells (data not shown), consistent with 1 acting as an uncoupler rather than stimulating ATP turnover. To further test the possibility that 1 uncouples, mitochondrial membrane potential was monitored with the fluorescent lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM+). Mitochondrial TMRM+ accumulation was substantially reduced by FCCP, but only modestly affected by 1 (Figure 2A), suggesting that 1 may only weakly uncouple. A
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In a human breast tumor T47D cell-based respiration study, 1 stimulated cellular oxygen consumption in a concentrationdependent manner (50% increase at 30 μM, Figure 1A). Similar respiration-enhancing effects were observed in the yeast S. cerevisiae (Figure 1B). To discern if 1 stimulated respiration by protonophore-based uncoupling or by stimulating cellular ATP consumption, the effect of 1 on oligomycin-induced state 4
Figure 1. Sampagine stimulates oxygen consumption in both metazoan and yeast cells. (A) The rate of oxygen consumption in intact T47D cells was determined in the presence and absence of compounds at the specified concentrations. Data were normalized to the untreated control and are presented as mean + range from two independent experiments. The mitochondrial uncoupler FCCP was included as a positive control. (B) The effect of 1 on oxygen consumption in the budding yeast S. cerevisiae was determined using Oxytherm, similar to that described in part A. Data shown are average + standard deviation from three independent determinations, and the asterisk indicates a statistically significant difference (p < 0.05) between the compound-treated samples and the untreated control. (C) Compound 1 overcame oligomycin-suppressed T47D cell respiration in a concentration-dependent manner. (D) Reinitiation of oligomycin-stalled cellular respiration by the standard uncoupler FCCP in T47D cells.
Figure 2. Sampangine acts as an atypical uncoupler. (A) Effect of 1 on mitochondrial membrane potential. T47D cells were loaded with the fluorescent cationic dye TMRM+. Microscopic pictures shown are live cell images before and after the addition of test compounds. The standard uncoupler FCCP was included as a positive control. (B) Quantitative results of 1 treatment on mitochondrial membrane potential and intracellular calcium concentration in T47D cells. TMRM fluorescence intensity was measured as an indicator of mitochondrial membrane potential, and the 340/380 nm excitation ratio in fura-2 loaded cells correlated with intracellular Ca2+ concentration. Data shown are average ± SD from the measurements of 20 cells. (C) Digitonin-permeabilized T47D cells were exposed to NaN3 (10 mM) to suppress mitochondrial respiration. Oxygen consumption was monitored to determine the impact of test compounds on mitochondrial substrate oxidation. Data shown are representative of two independent experiments. B
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time course-based quantitative study revealed that 1 marginally decreased TMRM+ fluorescence and did not affect the level of intracellular calcium that normally oscillates upon mitochondrial membrane potential alteration (Figure 2B). The fact that 1 increases oxygen consumption 1.5-fold (similar to FCCP) with minimal perturbation of mitochondrial TMRM accumulation suggests that 1 may primarily increase non-mitochondrial oxygen consumption. To explore this possibility, the effect of 1 on cellular oxygen consumption was determined in the presence of sodium azide, an electron transport chain complex IV inhibitor that suppresses mitochondrial oxygen consumption. Figure 3 depicts a generalized mitochondrial inhibitor-
Figure 4. Ascorbate augments 1-stimulated oxygen consumption. (A) Compound 1 reinitiated sodium azide-stalled oxygen consumption that is enhanced by ascorbate in a concentration-dependent manner. Oxygen consumption in T47D cells was monitored using a Clark-type electrode. T47D cells were permeabilized with digitoin, and the compounds were added in a sequential order at the specified final concentrations and time: NaN3 (10 mM), 1 (30 μM), solvent (2propanol, 0.6%), or ascorbate (Asc., 1 and 5 mM, respectively). Data shown are representative of at least two independent experiments. (B) Relative oxygen consumption. Oxygen consumption rate (OCR) was determined following compound addition, and the data were normalized to that of the NaN3. Data shown are mean ± range from two experiments.
Figure 3. Generalized mitochondrial inhibitor-based mechanistic investigation of a compound that induces oxygen consumption.
based mechanistic investigation of a compound that induces oxygen consumption. Compound 1 stimulated sodium azideinsensitive oxygen consumption nearly to the same extent as without azide [1 (30 μM)-stimulated oxygen consumption is equivalent to 46% of the control in the absence of sodium azide and 48% in the presence of sodium azide, Figure 2C], suggesting that much of the increase did not depend on complex IV and was thus non-mitochondrial. Compounds such as quinones and anthroquinones produce ROS through redox cycling, a process that can be enhanced by coupling the reaction with ascorbate oxidation/reduction.12 The effect of ascorbate on sampangine-induced oxygen consumption was examined using the T47D cell-based respiration assay. Ascorbate augmented 1-stimulated oxygen consumption in the presence of sodium azide (Figure 4A), and this enhancement was concentration-dependent (Figure 4B). In contrast, FCCP-increased oxygen consumption was not affected by ascorbate (Figure 4B). These results support the hypothesis that 1 stimulates non-mitochondrial oxygen consumption and ascorbate facilitates the quinone/anthraquinone-like redox cycling of 1 (Figure 6). As master regulators of oxygen homeostasis, hypoxiainducible factors (HIFs) mount cellular responses to decreased oxygen tension (hypoxia) at the transcription level.13,14 Composed of an oxygen-labile α subunit and a constitutively expressed β subunit (HIF1β/ARNT), HIF-1 is activated by hypoxic conditions.13,14 Mitochondria constitute an important module in the hypoxic signaling network that regulates HIF-1 activity.15−20 The effect of 1 on HIF-1 activity was examined in
a T47D cell-based reporter assay.19 A concentration-dependent biphasic response was observed (Figure 5A). Compound 1 enhanced hypoxic activation of HIF-1 at lower concentrations and inhibited it at higher concentrations. Mitochondriagenerated ROS are known to mediate hypoxic HIF-1 activation.21 It is possible that 1-stimulated ROS production enhanced hypoxic signaling at lower concentrations. At higher concentrations, 1-imposed cytotoxicity may contribute to the inhibition of HIF-1 activity (Figure 5A). In a standard 48 h exposure viability study, 1 displayed a more pronounced cytostatic/cytotoxic effect on estrogen-dependent T47D cells, in comparison to the triple-negative breast cancer (TNBC) MDA-MB-231 cells (Figure 5B). Ascorbate enhanced the growth inhibitory activity exerted by 1 (Figure 4B). The potential toxicity of 1 was further examined using rat cerebellar granule neurons (CGNs) as an in vitro model.22 Primary CGNs isolated from rat pups were maintained in culture for 6−10 days before compound treatment. After 24 h incubation with compounds, cells were stained with Hoescht 33342 and propidium iodide (PI). Representative images of untreated CGNs (control) and those exposed to 1 (30 μM) are shown in Figure 5C. Quantification of live (PI negative and healthy) and dead or dying (PI positive or PI negative with condensed or fragmented nuclei) cells was applied to determine the extent of cell death. Exposure to 1 killed all the neurons (Figure 5C). In summary, 1 primarily increased cellular oxygen consumption through a non-mitochondrial process that likely involves redox cycling of 1. Because 1 shares structural features C
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Figure 5. continued untreated CGNs (control) and those exposed to 1 (30 μM) are shown in the top panel. Four randomly selected fields for each condition were chosen to quantify the number of live (PI negative and healthy) and dead or dying (PI positive or PI negative with condensed or fragmented nuclei) cells. Data shown in the bottom panel are averages + standard deviation from one experiment, representative of two independent experiments.
Figure 6. Example of one cycle in ascorbate-coupled redox cycling of sampangine to generate biologically active ROS. Similar cycling to generate ROS can be made through coupling the reaction with mitochondrial complex I.
with the electron carrier ubiquinone, it is possible that this compound is reduced to a reactive semi-iminoquinone form by mitochondria (e.g., complex I) that then readily reduces oxygen to superoxide. In support of this scheme, ascorbate, an electron donor for a variety of compounds,12 readily increased azideinsensitive respiration only in the presence of 1 and also potentiated T47D cell death (Figure 5B), effects consistent with redox cycling and ROS production. These results are consistent with Bailly and co-workers’ previous observation of a sampangine-induced ROS burst and ROS-associated apoptosis in HL-60.6 Similar mechanisms have also been attributed to the cytotoxic effects produced by the marine iminoquinone moiety of the marine tunicate heterocyclic compounds known as ascididemnins.23 Along this line, recent attempts to improve the therapeutic potential of 1 by structure modification have produced thiophone-quinones that retain much of the antifungal and cytotoxic activity, but no longer require the complexity of an iminoquinone heterocycle.3 Sampangineinduced redox cycling and its associated ROS-induced oxidative stress may interfere with cellular signaling pathways such as those that regulate HIF-1 activation and produce a neurotoxic response in rat cerebellar granule neurons. The precise etiology for the observed effects of 1 on heme biosynthesis10 and uroporphyrinogen III synthase11 has not been determined. Heme is an important component of cytochromes, which are present in complexes III and IV of the electron transport chain, and it is difficult to rule out the possible role of heme dysfunction in 1-induced ROS production, although it is well established that heme-associated iron and various heme biosynthetic intermediates are known to be oxidized via reactions with cellular ROS.26 For example, heme biosynthesis can be coupled to the mitochondrial electron transport chain
Figure 5. Sampangine regulates HIF-1 activation and inhibits cell proliferation/viability. (A) Sampangine displays a concentrationdependent biphasic effect on HIF-1 activity. T47D cells transfected with the pHRE-luc construct were exposed to hypoxic conditions (1% O2, 16 h) in the presence of emetine (positive control) or 1 at the specified concentrations. Luciferase activity and cell viability were determined and presented as “% Inhibition” of the media control under hypoxia. Data shown are average ± SD (n = 3). (B) Concentration−response of 1 on T47D and MDA-MB-231 cell proliferation/viability, in the presence and absence of ascorbate (1 mM). After 48 h compound treatment, cell viability was determined by the SRB method and presented as % Inhibition of the media control. Data shown are average + SD (n = 3). (C) Representative images of D
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for energy production,27 oxidative stress disregulates heme production,28 ROS induced by the herbicide paraquat disrupts heme biosynthesis,29 and ROS suppresses uroporphyrinogen III synthase gene expression.30 Redox cycling appears responsible for the observed immediate increase of cellular oxygen consumption, but defects in heme biosynthesis may disrupt an array of biochemical processes. While these studies help illustrate the regulator pathways involved in the mechanism of action of 1, they also suggest that ROS-mediated side effects and toxicity may significantly limit the clinical potential of sampangine and its analogues as antifungal and antitumor agents.
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Data Analysis. GraphPad Prism 6 was applied to process data. Differences between data sets were considered statistically significant when p < 0.05.
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AUTHOR INFORMATION
Corresponding Authors
*Tel: (662) 915-2215. Fax: (662) 915-7300. E-mail: ydzhou@ olemiss.edu (Y.-D. Zhou). *Tel: (662) 915-7026. Fax: (662) 915-6975. E-mail: dnagle@ olemiss.edu (D. G. Nagle). Notes
The authors declare no competing financial interest.
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EXPERIMENTAL SECTION
ACKNOWLEDGMENTS The authors thank Dr. S. L. McKnight (University of Texas Southwestern Medical Center at Dallas) for providing the pHRE3-TK-luc construct and Dr. D. Ferreira (University of Mississippi) for mechanistic discussions and advice. This work was supported in part by the National Cancer Institute, NIH (grant CA98787). Support for W.L. was provided by the China Scholarship Council. This investigation was conducted in a facility constructed with Research Facilities Improvement Grant C06 RR-14503 from the NIH.
General Experimental Procedures. Human breast tumor T47D, MDA-MB-231, and hepatoma Hep3B cells (ATCC, Manassas, VA, USA) were maintained in DMEM/F12 media with L-glutamine (Mediatech, Manassas, VA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 50 units/mL penicillin, and 50 μg/mL streptomycin (Gibco, Grand Island, NY, USA). Sampangine was prepared as previously reported4 and determined to be greater than 99% pure by analytical RP-HPLC (percentage of the integrated signal at UV 254 nm). Unless specified, all other chemicals were purchased from Sigma (St. Louis, MO, USA). Cell-Based Respiration Assay. An Oxytherm Clark electrode system (Hansatech, Norfolk, UK) was used to monitor oxygen consumption by T47D, Hep3B, and S. cerevisiae cells. The experimental procedure was similar to that previously reported.19,24,25 The following formula was used for data presentation:
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