NADH Ratio Are

Article pubs.acs.org/JAFC

Sirtuin 1 (SIRT1) Deacetylase Activity and NAD+/NADH Ratio Are Imperative for Capsaicin-Mediated Programmed Cell Death Yi-Hui Lee,† Huei-Yu Chen,† Lilly J. Su,†,Δ and Pin Ju Chueh*,†,§,#,⊥ †

Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan Graduate Institute of Basic Medicine, China Medical University, Taichung 40402, Taiwan # Department of Medical Research, China Medical University Hospital, Taichung 40402, Taiwan ⊥ Department of Biotechnology, Asia University, Taichung 41354, Taiwan §

ABSTRACT: Capsaicin is considered a chemopreventive agent by virtue of its selective antigrowth activity, commonly associated with apoptosis, against cancer cells. However, noncancerous cells possess relatively higher tolerance to capsaicin, although the underlying mechanism for this difference remains unclear. Hence, this study aimed to elucidate the differential effects of capsaicin on cell lines from lung tissues by addressing the signal pathway leading to two types of cell death. In MRC-5 human fetal lung cells, capsaicin augmented silent mating type information regulation 1 (SIRT1) deacetylase activity and the intracellular NAD+/NADH ratio, decreasing acetylation of p53 and inducing autophagy. In contrast, capsaicin decreased the intracellular NAD+/NADH ratio, possibly through inhibition of tumor-associated NADH oxidase (tNOX), and diminished SIRT1 expression leading to enhanced p53 acetylation and apoptosis. Moreover, SIRT1 depletion by RNA interference attenuated capsaicin-induced apoptosis in A549 cancer cells and autophagy in MRC-5 cells, suggesting a vital role for SIRT1 in capsaicin-mediated cell death. Collectively, these data not only explain the differential cytotoxicity of capsaicin but shed light on the distinct cellular responses to capsaicin in cancerous and noncancerous cell lines. KEYWORDS: apoptosis, autophagy, capsaicin, silent mating type information regulation 1 (sirtuin 1, SIRT1), tumor-associated NADH oxidase (tNOX, ENOX2)

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cellular context and type.25−27 Capsaicin was recently reported to induce ER stress-mediated autophagy/apoptosis through activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in normal lung epithelial fibroblast WI-38 cells.28 Similarly, both autophagy and apoptosis were observed in capsaicin-exposed human osteosarcoma G292 cells, and inhibition of autophagy appeared to enhance apoptosis.29 Interestingly, 250 μM capsaicin exposure is found to trigger autophagy in malignant MCF-7 and MDAMB-231 breast cells, leading to enhanced chemoresistance and prolonged cancer cell survival.30,31 These diverging cellular responses place capsaicin in the pivotal position of differentially modulating pathways that lead to either cell death or survival. This dual effect of capsaicin on cell growth creates an urgent need to identify molecular pathways that are viable targets for cancer management. On account of the above findings, we used MRC-5 normal human lung tissue fibroblast and A549 human lung adenocarcinoma epithelial cell lines to investigate the differential effects of capsaicin and the underlying mechanisms for this difference. Capsaicin is found to preferentially inhibit the tumor-associated NADH oxidase (tNOX) activity and cytotoxic apoptosis of transformed cells,1 which normally oxidizes hydroquinones and NADH, converting the latter to the

INTRODUCTION Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is an important constituent of chili peppers. Because of its anti-growth activity against a range of cancer cell systems,1−5 capsaicin has been used as a chemoprevention agent. In most cases, cancer cell death by apoptosis is associated with capsaicin-mediated cytotoxicity through activation of numerous mechanisms, including generation of reactive oxygen species (ROS),6−8 initiation of endoplasmic reticulum (ER) stress,9,10 and alteration of protein kinases.11,12 More importantly, accumulating evidence suggests that capsaicin induces apoptosis selectively in transformed/cancer cells but not in noncancer lines,1,13−15 further enhancing the application of capsaicin in chemoprevention. However, the fundamental molecular mechanisms underlying this differential action remain unclear. Apoptosis and autophagy both are well-regulated processes involved in many important cellular functions, although in most cases apoptosis represents a self-destruct mechanism as opposed to the cytoprotective effect of autophagy.16−18 The extrinsic pathway of apoptosis is initiated by binding to death receptors, which subsequently activates initiator caspase-8 and effector caspases.19 Alternatively, the intrinsic pathway is dependent on the translocation of Bax/Bak to mitochondria and release of cytochrome c from mitochondria into the cytoplasm leading to subsequent caspase cascade and degradation of the cell.20−24 Autophagy is a self-digestion and bulk-degradation process, with adaptive catabolic and energygenerating features that promote cellular survival in response to stresses; nevertheless, it may also be cytotoxic, depending on © XXXX American Chemical Society

Received: June 10, 2015 Revised: August 7, 2015 Accepted: August 8, 2015

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DOI: 10.1021/acs.jafc.5b02876 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry oxidized NAD+ form.32,33 Thus, it is of interest to study whether tNOX and the NAD+/NADH ratio are involved in the differential effect of capsaicin. Here, we present evidence for the first time that the intracellular NAD+/NADH ratio and SIRT1 deacetylase activity are central to the regulation of capsaicininduced cell death.

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exposed to capsaicin or ethanol, and autophagosomes formation was visualized. Measurement of Intracellular NAD+/NADH Ratio. Both oxidized and reduced forms of intracellular nicotinamide adenine dinucleotide were determined using an NADH/NAD Quantification Kit (BioVision Inc., Milpitas, CA, USA). Briefly, 2 × 105 cells were washed with cold PBS, pelleted, and extracted with 400 μL of NADH/ NAD extraction buffer by two freeze/thaw cycles. Samples were vortexed and centrifuged at 14000 rpm for 5 min. For each sample, a 200 μL aliquot of extracted NADH/NAD supernatant was transferred to a microcentrifuge tube. Samples were first heated to 60 °C for 30 min to allow decomposition of all NAD+, while retaining NADH intact, and then placed on ice. Samples were rapidly centrifuged and transferred to a multiwall plate. Standards and a NAD cycling mix were prepared according to the manufacturer’s protocol. One hundred microliters of the reaction mix was added into each well of NADH standards and samples, and all samples were incubated at room temperature for 5 min to convert NAD+ to NADH. NADH Developer Solution was added to each well, and plates were incubated at room temperature for 30 min. The reaction was stopped by adding 10 μL of Stop Solution, and absorbance was measured at 450 nm. Measurement of SIRT1 Deacetylase Activity in Vitro. SIRT1 deacetylase activity was determined using a SIRT1 direct Fluorescent Screening Assay Kit (Cayman Chemical Co., Ann Arbor, MI, USA). Briefly, the substrate solution was first prepared by adding 240 μL of NAD+ solution and 850 μL of diluted assay buffer to 15 μL of the p53 peptide Arg-His-Lys-Lys(ε-acetyl)-AMC, to yield final concentrations of 125 μM peptide (substrate) and 3 mM NAD+. Background was determined in wells containing 30 μL of assay buffer and 5 μL of solvent. Maximal initial activity (defined as 100%) was determined in wells containing 5 μL of diluted human recombinant SIRT1, 25 μL of assay buffer, and 5 μL of solvent. For our experiments, 5 μL of capsaicin was added to wells containing 25 μL of assay buffer and 5 μL of diluted human recombinant SIRT1. Reactions were initiated by adding 15 μL of substrate solution to all of the wells. The plate was then covered and incubated on a shaker for 45 min at room temperature. Reactions were stopped by adding 50 μL of Stop/ Developing Solution to each well and incubating the plate for 30 min at room temperature. Plates were read in a fluorometer using an excitation wavelength of 350−360 nm and an emission wavelength of 450−465 nm. Western Blot Analysis. Cell extracts were prepared in lysis buffer containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 2 mM phenylmethanesulfonyl fluoride (PMSF), 10 ng/mL leupeptin, and 10 μg/mL aprotinin. Volumes of extract containing equal amounts of proteins (40 μg) were applied to SDS-PAGE gels, and resolved proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). The membranes were blocked with nonfat milk solution for 30 min, then washed, and probed with primary antibody. Membranes were then rinsed with Tris-buffered saline containing 0.1% Tween 20 to remove unbound primary antibody and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h. The membranes were rinsed again and developed using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Piscataway, NJ, USA). Statistics. All data are expressed as the mean ± SE of three or more independent experiments. Comparison between groups was made by one-way analysis of variance (ANOVA) followed by an appropriate post hoc test to analyze the difference. A value of p < 0.05 was considered to be statistically significant.

MATERIALS AND METHODS

Cell Culture and Reagents. Capsaicin with purity >95% was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Anti-Bax, anti-Bak, anti-PARP, anti-Bcl-2, anti-p53, anti-acetyl-p53, anti-SIRT1, anti-mTOR, anti-phospho-mTOR, anti-phospho-AMPK, anti-phospho-JNK, and anti-acetyl-Lys antibodies were from Cell Signaling Technology, Inc. (Beverly, MA, USA). Anti-beclin-1, anti-Atg5, antiAtg7, and anti-LC3 antibodies were from Novus Biologicals, Inc. (Littleton, CO, USA). The anti-survivin antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-β-actin antibody was from Millipore Corp. (Temecula, CA, USA). Antisera to tNOX were generated as described previously.34Other chemicals were from Sigma-Aldrich Corp. A549 (human lung adenocarcinoma epithelial) cells were grown in DMEM, and MRC-5 (human lung tissue) cells were grown in MEM. All media were supplemented with 10% FBS, 100 U/mL penicillin, and 50 μg/mL streptomycin. Cells were maintained at 37 °C in a 5% CO2 −95% air-humidified incubator. ON-TARGETplus tNOX (ENOX2) siRNA and negative control siRNA were purchased from Thermo Scientific, Inc. (Grand Island, NY, USA), and SignalSilence Sirt1 siRNA I and control siRNA were from Cell Signaling Technology, Inc. Briefly, cells were seeded in 10 cm dishes and allowed to attach overnight. The next day, cells were transfected with tNOX/Sirt1 siRNA and control siRNA using Lipofectamin RNAiMAX reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions.35 Cell Impedance Measurements. Cell impedance technology was used to continuously monitor changes in cell growth. Cells (104 cells/ well) were seeded onto E-plates. After 30 min of incubation at room temperature, E-plates were then set down onto the Real-Time Cell Analysis station (Roche, Mannheim, Germany). Cells were grown overnight before exposure to ethanol or different concentrations of capsaicin, and cell impedance was measured every hour, as previously described.36 WST-1 Cell Viability Assay. Cells (5 × 103) were seeded in 96well culture plates and permitted to adhere overnight. Cells were then treated with capsaicin or ethanol for 72 h, and at the end of treatment, cell viability was determined using a WST-1 assay (Roche Applied Science, Mannheim, Germany). Apoptosis Determination. Annexin V-FITC Apoptosis Detection Kits were used to determine apoptosis (BD Pharmingen, San Jose, CA, USA). Briefly, cells treated with ethanol or different concentrations of capsaicin were harvested by centrifugation after trypsinization. Cell pellet was washed with PBS and resuspended in 1× binding buffer. Next, cell pellet was stained with annexin V-FITC (fluorescein isothiocyanate) and also propidium iodide (PI) according to the manufacturer’s protocol. The patterns of cell death (necrosis and apoptosis) were analyzed using a Beckman Coulter FC500 flow cytometer, and results were expressed as a percentage of total cells. Autophagy Determination. Autophagosomes were visualized by staining with Acridine Orange (AO). After incubation, cells were washed with PBS and stained with 1 mg/mL AO for 15 min at 37 °C. AO-stained cells were then washed, trypsinized, and analyzed using a Beckman Coulter FC500. The results are expressed as a percentage of total cells. Visualization of Autophagosome Formation Using EGFPLC3. For autophagy experiments, MRC-5 cells were transiently transfected with a pEGFP-LC3 construct (a generous gift from Dr. Tamotsu Yoshimori, Osaka University, Japan) using Lipotectamine 2000 reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).37 MRC-5-EGFP-LC3 cells were either starved or

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RESULTS A549 Cells Exhibit Greater Responsiveness to Capsaicin than MRC-5 Cells. Given that transformed and noncancerous cells exhibit differential cytotoxicity to capsaicin, we decided to choose lines from lung tissue, MRC-5 (human lung tissue) and A549 (human lung adenocarcinoma), cells in this study. We continuously monitored the dynamic effects of capsaicin on cell growth, measuring cell impedance and B


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Journal of Agricultural and Food Chemistry

Figure 1. Capsaicin inhibits cell growth in A549 cells but not in MC-5 cells. (A, B) Cell growth with or without capsaicin was dynamically monitored using impedance technology, in MRC-5 cells (A) and in A549 cells (B). Normalized cell index values measured over 108 h are shown. (C, D) Cells were treated with capsaicin for 72 h, and cell viability was determined by WST-1 assay, in MRC-5 cells (C) and in A549 cells (D). Values (mean ± SE) are from three independent experiments performed in at least triplicates: (∗) p < 0.05 and (∗∗) p < 0.01 for A549 cells treated with capsaicin versis controls.

displaying the results as cell index values.38 Using this approach, we found that MRC-5 cell growth was decreased by capsaicin at an early stage of exposure; however, cell growth gradually recovered, resulting in little overall inhibition at all concentrations tested (Figure 1A). In contrast, capsaicin induced a significant concentration-dependent decrease in cell growth in A549 cells; this inhibitory effect was much more evident than that in MRC-5 cells (Figure 1B). The greater responsiveness of A549 cells to capsaicin is reflected in IC50 values, which was determined to be 33.8 μM for A549 cells but could not be calculated in MRC-5 cells because the degree of inhibition was

so low in these cells. We also tested cell viability using WST-1 assays and demonstrated that the viability of MRC-5 cells was not inhibited significantly by capsaicin (Figure 1C), whereas that of A549 cells was decreased considerably (to less than 76 and 60% of control values at 100 and 200 μM, respectively) (Figure 1D). The IC50 value for capsaicin in A549 cells determined on the basis of WST-1 assays was 146 μM and could not be calculated for MRC-5 cells due to a low degree of inhibition. These data suggested that capsaicin is cytotoxic in A549 cells but not in MRC-5 cells. C


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Figure 2. Capsaicin preferentially induces autophagy but not apoptosis in MRC-5 cells. (A) MRC-5 cells were treated with capsaicin or ethanol for 24 h. The percentage of apoptotic cells was determined by flow cytometry, and the results are expressed as a percentage relative to the control group. Values (mean ± SE) are from three independent experiments. No significant differences were observed. (B) Cells were treated with capsaicin or ethanol for 6 h. Autophagy was determined by AO staining using flow cytometry, and the results are expressed as a percentage relative to the control group. Values (mean ± SE) are from four independent experiments: (∗) p < 0.05 for MRC-5 cells treated with 200 μM capsaicin versus controls. (C) MRC-5 cells were pretreated with 0.1 μM BafA1 or not for 30 min before exposure to capsaicin for 6 h. The percentage of autophagic cells was assessed by flow cytometry, and the results are expressed as a percentage relative to the control group. Values (mean ± SE) are from three independent experiments: (∗) p < 0.05 for MRC-5 cells without BafA1 pretreatment treated with capsaicin versus controls. (D) MRC-5 cells were transiently transfected with EGFP-LC3 for 48 h and serum starved or treated with capsaicin or ethanol for 6 h. EGFP fluorescence was observed under a fluorescence microscopy. (E, F) MRC-5 cells were treated with capsaicin or ethanol for 24 h. Aliquots of cell lysates were separated by SDSPAGE and analyzed by Western blotting; β-actin was used as internal control. Representative images from three experiments are shown. (G) MRC-5 cells were pretreated with 100 μM 3-MA or not for 1 h before exposure to capsaicin or ethanol for 24 h. The percentage of apoptotic cells was assessed by flow cytometry, and the results are expressed as a percentage relative to the control group. Values (mean ± SE) are from four independent experiments: (∗) p < 0.05 for MRC-5 cells without 3-MA pretreatment versus other groups.

Capsaicin Induces Autophagy in MRC5 Cells but Not Apoptosis. To determine whether programmed cell death is involved in the differential cellular responses to capsaicin, we analyzed cells for apoptotic subpopulations. Interestingly, little apoptotic effect was observed in capsaicin-exposed MRC-5 cells, indicating that MRC-5 cells were largely impervious to the apoptotic effect of capsaicin (Figure 2A). We also explored the possibility that capsaicin induces autophagya type II programmed cell death-related pathwayin these cells. Using AO staining for detection of acidic vesicular organelles, we found that 200 μM capsaicin significantly increased the population of MRC-5 cells undergoing autophagy at 6 h (Figure 2B). Pretreatment of MRC-5 cells with the autophagy inhibitor bafilomycin A1 (BafA1) has shown to reverse the capsaicin-induced autophagy (Figure 2C). To further explore autophagy in capsaicin-exposed MRC-5 cells, we exogenously expressed a fusion protein of enhanced green fluorescent protein and the autophagy marker microtubule associated light chain 3 protein (EGFP-LC3) and observed the fluorescence. We showed that capsaicin exposure resulted in autophagosomes formation as green punctuate dots, similar to that observed in serum-starved cells (Figure 2D). Moreover, protein analyses verified that important autophagy regulators, such as autophagy-related 7 (Atg7), Atg5−Atg12 conjugate, beclin-1, and cleaved LC3 II, were up-regulated, whereas phosphorylated and total mammalian target of rapamycin (mTOR) were down-

regulated by capsaicin (Figure 2E). Expressions of apoptosisrelated proteins also indicated a lack of apoptosis in capsaicinexposed MRC-5 cells, pro-apoptotic Bak protein Bax were decreased, pro-survival Bcl-2 protein was increased, and a caspase-3-mediated poly(ADP-ribose) polymerase (PARP) cleavage was not detected (Figure 2F). Pretreatment of MRC-5 cells with the autophagy inhibitor 3-methyladenine alone indeed significantly enhanced apoptosis; however, it had little impact on capsaicin-induced apoptosis (Figure 2G). Capsaicin Induces Cytotoxic Apoptosis in A549 Cells. To verify that transformed and noncancerous cells indeed exhibit differential cytotoxicity to capsaicin, lung carcinoma cells were used in this study. In A549 cells, a 24 h capsaicin exposure induced a significant, >2-fold, increase in apoptosis at 200 μM (Figure 3A). Western blot analyses also revealed that capsaicin triggered considerable up-regulation of the Bak and Bax as well as cleaved PARP, with concurrent down-regulation of Bcl-2 and survivin in capsaicin-exposed A549 cells (Figure 3B). However, we found a lack of capsaicin-induced autophagy at 6 h of exposure (Figure 3C). Changes in autophagy protein levels were less marked in A549 cells (Figure 3D). Moreover, pretreatment with z-VAD, a pan-caspase inhibitor, had little effect on capsaicin-induced autophagy (Figure 3E). Capsaicin Modulates Intracellular NAD+/NADH Ratio. An association between capsaicin-inhibited tumor-associated NADH oxidase (tNOX) activity and cytotoxic apoptosis has D


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Figure 3. Capsaicin induces apoptosis but not autophagy in A549 cells. (A) A549 cells were treated with capsaicin or ethanol for 24 h. The percentage of apoptotic cells was determined by flow cytometry, and the results are expressed as a percentage relative to the control group. Values (mean ± SE) are from four independent experiments: (∗∗) p < 0.01 for A549 cells treated with 200 μM capsaicin versus controls. (B, D) A549 cells were treated with capsaicin or ethanol for 6 or 24 h. Aliquots of cell lysates were separated by SDS-PAGE and analyzed by Western blotting. β-Actin was used as internal control. Representative images from three experiments are shown. (C) A549 cells were treated with capsaicin or ethanol for 6 h. Autophagy was determined by AO staining using flow cytometry, and the results are expressed as a percentage relative to the control group. Values (mean ± SE) are from three independent experiments: (∗) p < 0.05 and (∗∗) p < 0.01 for A549 cells treated with starvation or 200 μM capsaicin versus controls. (E) A549 cells were pretreated with 100 μM z-VAD for 1 h before exposure to capsaicin or ethanol for 6 h. The percentage of autophagic cells was assessed by flow cytometry, and the results are expressed as a percentage relative to the control group. Values (mean ± SE) are from three independent experiments. There were no significant differences in this experiment.

Figure 4. Modulation effects of capsaicin and tNOX-knockdown on cellular redox status and protein expression. (A) Cells were treated with ethanol or capsaicin, and NAD+ and NADH in cell extracts were then quantified. The optical density at 450 nm was recorded and used to calculate the NAD+/NADH ratio. Values (mean ± SE) are from three independent experiments: (∗) p < 0.05 for cells treated with 200 μM capsaicin versus others. (B) Cells were treated with capsaicin or ethanol for 24 h. Aliquots of cell lysates were separated by SDS-PAGE and analyzed by Western blotting; β-actin was used as internal control. Representative images from three experiments are shown. (C) tNOX was down-regulated using siRNA. Aliquots of cell lysates were separated by SDS-PAGE and analyzed by Western blotting; β-actin was used as internal control. Representative images from three experiments are shown. (D) NAD+ and NADH in control and tNOX-depleted A549 cell extracts were quantified. The optical density at 450 nm was recorded and used to calculate the NAD+/NADH ratio. Values (mean ± SE) are from three independent experiments: (∗) p < 0.05 for tNOX-depleted cells versus controls. E


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Figure 5. Modulation effects of capsaicin on SIRT1 in vitro activity and protein expression. (A) Samples were incubated with ethanol or capsaicin, and SIRT1 deacetylase activity in vitro was determined using a direct fluorescence assay kit. Nicotinamide was used as a control. Values (mean ± SE) are from three independent experiments performed in at least triplicates: (∗∗) p < 0.01 and (∗) p < 0.05 for samples treated with capsaicin or nicotinamide versus controls. (B, C) Cells were treated with capsaicin or ethanol for 24 h, in MRC-5 cells (B) and in A549 cells (C). Aliquots of cell lysates were separated by SDS-PAGE and analyzed by Western blotting; β-actin was used as internal control. Representative images from three experiments are shown.

Figure 6. Effect of SIRT1 depletion on capsaicin-induced programmed cell death. (A) SIRT1 was down-regulated by siRNA, and cells were treated with ethanol or capsaicin. Aliquots of cell lysates were separated by SDS-PAGE and analyzed by Western blotting; β-actin was used as internal control. Representative images from three experiments are shown. (B) Control or SIRT1-depleted MRC-5 cells were treated with capsaicin or ethanol for 6 h. Autophagy was determined by AO staining using flow cytometry, and the results are expressed as a percentage relative to the control group. Values (mean ± SE) are from four independent experiments: (∗) p < 0.05 for siRNA-control cells versus SIRT1-knockdown controls and also for siRNA-control cells treated with 200 μM capsaicin versus untreated controls. (C) Control and SIRT1-depleted A549 cells were treated with capsaicin or ethanol for 24 h. Apoptosis was determined by annexin V staining by flow cytometry, and the results are expressed as a percentage of apoptotic cells. Values (mean ± SE) are from three independent experiments: (∗) p < 0.05 for siRNA-control cells versus SIRT1-knockdown controls and also for siRNA-control cells treated with 200 μM capsaicin versus untreated controls.

cells (Figure 4A). Treatment of A549 cells with 200 μM capsaicin significantly decreased the NAD+/NADH ratio but increased it in MRC-5 cells, although the change was relatively small compared with that in A549 cells (Figure 4A). These data support that tNOX oxidizes NADH to NAD+ and capsaicininhibited tNOX resulted in a decrease in the intracellular NAD+/NADH ratio in cancer cells. Moreover, capsaicin (200 μM) down-regulated tNOX expression in A549 cells, whereas tNOX expression was not detected in MRC-5 cells (Figure 4B), confirming tNOX expression in a cancerous, but not a nontransformed, line. We further found that targeting tNOX in A549 cells with siRNA (Figure 4C) decreased the NAD+/

been previously established only in cancer/transformed cells.1 This observation prompted us to examine whether the capsaicin-induced cytotoxicity in A549 cells involves tNOX and, if so, by what mechanism. NAD+ is a biological oxidizing agent for many metabolic reactions, and tNOX oxidizes hydroquinones and NADH, converting the latter to the oxidized NAD+ form.33 Thus, it is reasonable to presume that a capsaicin-inhibited tNOX could potentially disturb cellular redox homeostasis and, consequently, disrupt cellular responses. In this regard, we found that the basal NAD+/ NADH ratio in A549 cells was much greater than that in MRC5 cells, possibly reflecting endogenous tNOX activity in A549 F


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Figure 7. Schematic diagram of the mechanism of capsaicin-induced programmed cell death.

pathways. Transient receptor potential vanilloid type 1 (TRPV1) is the most-often mentioned protein targets of capsaicin that belongs to a member of the TRP family of nonselective cation channels.40 Several studies have suggested that capsaicin exerts its anticancer actions through interaction with the TRPV1, although capsaicin also acts through TRPV1 as a cocarcinogen.41,42 Here, we present evidence demonstrating that capsaicin modulates SIRT1 deacetylase activity; in particular, depending on the cellular context, capsaicin decreases the NAD + /NADH ratio and increases p53 acetylation, resulting in enhanced cytotoxic apoptosis (A549 cells), or increases the NAD+/NADH ratio and SIRT1 deacetylase activity toward p53, leading to survival autophagy (MRC-5 cells). These pathways are summarized in Figure 7. One major finding of this study is our clarification of the molecular mechanisms underlying the differential effect of capsaicin on cancer and noncancer cells. Capsaicin has long been considered as a chemopreventive agent by virtue of its inhibitory effect on cancer cell survival and proliferation as well its induction of apoptosis.15,43,44 Interestingly, noncancerous cells exhibit greater tolerance to capsaicin compared to cancer cells, although the basis for this selective cytotoxicity is uncertain.15 Consistent with the reports of others, our study provides support for the idea that capsaicin induces different mechanisms leading to distinct outcomes depending on cell type and cellular context.1,4,14 Our results confirmed that capsaicin preferentially induces cytotoxicity in A549 human lung cancer cells but not in MRC-5 cells normal human lung tissue, as supported by the observed IC50 value of 33.5 μM (based on cell impedance measurements) for A549 cells, but unmeasurable IC50 value in MRC-5 cells. A similar phenomenon was also reported in H-ras-transformed MCF10A and their parental noncancerous MCF10A cells.14 The current study further demonstrated that capsaicin acts through SIRT1 to selectively induce two types of programmed cell death-like pathways: protective autophagy in noncancerous cells but cytotoxic apoptosis in cancer cells. Another significant finding of this study is the demonstration of a regulatory role of SIRT1 in capsaicin-induced programmed cell death. SIRT1 belongs to the sirtuin protein family of NAD+-dependent deacetylases that target histones and nonhistone proteins.45,46 To our knowledge, Lee et al.47 is first to focus on the links between SIRT1 and capsaicin. Although these researchers found that capsaicin acted through inhibition of apoptosis to exert a neuroprotective effect against glutamateinduced toxicity, they found no significant effect of capsaicin on SIRT1 mRNA levels. Thus, it remained unclear whether capsaicin affected SIRT1 deacetylase activity and its downstream target proteins. In this study, we found that capsaicin reduced SIRT1 expression in A549 cells (Figure 5B), although

NADH ratio compared to A549 cells expressing control (scrambled) siRNA (Figure 4D). We also noted that tNOXdepleted A549 cells exhibited reduced silent mating type information regulation 1 (SIRT1) expression, whereas p53 acetylation and JNK phosphorylation were increased and cyclin D was attenuated (Figure 4C), confirming a tumor-promoting role of tNOX in cancer cells. Capsaicin Inhibits SIRT1 Deacetylase Activity in A549 Cells but Enhances It in MRC-5 Cells. Given that NAD+ is also a cofactor for SIRT1 deacetylase, we were interested in determining whether the opposing effects of capsaicin on the NAD+/NADH ratio in A549 and MRC-5 cells affected SIRT1 activity. Utilizing an in vitro assay with recombinant SIRT1, we found that capsaicin effectively stimulated its activity at concentrations of 10−200 μM, whereas nicotinamide, a SIRT1 inhibitor, significantly decreased SIRT1 activity in vitro (Figure 5A). We also found that 200 μM capsaicin had little effect on SIRT1 expression in MRC-5 cells (Figure 5B). Thus, in the absence of a change in SIRT1 expression, the higher in vitro SIRT1 activity by capsaicin (Figure 5A) together with the capsaicin-induced increased in intracellular NAD+/ NADH ratio (Figure 4A) led to enhanced SIRT1 deacetylase activity. This interpretation is supported by the decrease in the acetylation level of p53 and enhancement of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation/activation, an activator of SIRT1 (Figure 5B). It is documented that deacetylation of p53 induces autophagy,39 and we also found that capsaicin reduced p53 acetylation accompanying the up-regulation of Atg5, Atg7, becline-1, cleaved LC3 II, and autophagy (Figure 2). Interestingly, exposure of A549 cells to 200 μM capsaicin reduced SIRT1 protein expression (Figure 5C). Alongside the decrease in intracellular NAD+/NADH ratio in capsaicin-exposed A549 cells (Figure 4A), we found that the p53 acetylation level was noticeably augmented (Figure 5C), possibly by the decreased SIRT1 acetylase activity. Thus, capsaicin induced acetylated/ activated/phosphorylated p53 (Figure 5C), and, in turn, upregulated apoptotic Bax and Bak; this, together with downregulation of Bcl-2, resulted in apoptosis in A549 cells (Figure 3). Furthermore, small interfering RNA (siRNA) effectively decreased SIRT1 expression in both lines (Figure 6A). In SIRT1-depleted MRC-5 cells, capsaicin-induced autophagy was diminished (Figure 6B), whereas capsaicin-induced apoptosis was reduced in SIRT1-knockdown A549 cells (Figure 6C), demonstrating a key role for SIRT1 in the regulation of capsaicin-mediated programmed cell death-related pathways.

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DISCUSSION Capsaicin acts on an array of cellular targets, several of which have been identified, and initiates a number of signaling G


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on the dose.59 For example, capsaicin functions as a cocarcinogen through EGFR signaling in the TPA-induced skin cancer model.60 Erin et al. also reported that capsaicin promotes a more aggressive gene expression phenotype and represses expression of pro-apoptotic proteins in breast cancer cells,61 supporting the idea that capsaicin acts on other targets to activate unanticipated pathways, subsequently leading to tumorigenesis. A study from our own laboratory has shown that low concentrations of capsaicin up-regulate tNOX expression in colon cancer cells in conjunction with enhanced cell proliferation and migration.62 Consistent with our findings, Waning et al. also present a precedent for the reinforcing effect of capsaicin on cell migration.63 Much remains to be learned regarding the cellular targets of capsaicin and the molecular mechanisms initiated by capsaicin that mediate its effects, from cell death to sustained cell survival. We have elucidated the differential effects of capsaicin utilizing cell lines from lung tissues addressing not only the role of tNOX in cancer cells but also the signal pathways leading to two types of programmed cell death. Our results demonstrated, for the first time, that intracellular NAD+/NADH ratio and SIRT1 deacetylase activity are central to the regulation of capsaicin-induced programmed cell deaths in cancer and noncancerous cell lines, specifically, apoptosis in A549 cancer cells but survival autophagy in MRC-5 cells. The findings of this study assist us in understanding the biological function of capsaicin and should provide a rational framework for the further development of improved chemopreventive strategies.

we have not examined whether capsaicin-induced SIRT1 downregulation is at the translational or transcriptional level at this point. It is suggested that p53 enhances microRNA-34a expression and promotes apoptosis48 and that SIRT1 is one of the direct target genes of microRNA-34a.49 Consistent with our results (Figure 5C), it is possible that capsaicin induces p53 activation, leading to microRNA-34a expression and SIRT1 down-regulation in A549 cells. The lack of inhibitory effect of capsaicin on SIRT1 expression in MRC-5 cells was perhaps due to less activation of p53 (Figure 5B); however, it remains unconfirmed at this point. Interestingly, activation of AMPK, an important activator for SIRT1, was found to be associated with capsaicin-induced apoptosis in cancer cells.11,50 However, whether capsaicininduced AMPK activation modulates SIRT1 activity and subsequent apoptosis was not examined further in these previous studies. A most recent report has demonstrated that capsaicin increases FOXO-1 acetylation through the modulation of SIRT deacetylase activity, leading to apoptosis in pancreatic cancer cells.51 All of this evidence provides a rationale to explore the role of SIRT1 in capsaicin-induced programmed cell death, as shown in our present study; capsaicin modulates SIRT1 deacetylase activity, leading to apoptosis in A549 cancer cells but autophagy in MRC-5 cells. By dissecting the different pathways triggered by capsaicin, we present evidence that the intracellular NAD+/NADH ratio contributes to the divergent effects of capsaicin in noncancer and cancer cell lines. NAD+ plays an essential role in many metabolic reactions, and it has recently emerged that NAD+ acts as a metabolic regulator, in part, through regulation of SIRT family.52 Given the metabolic centrality of NAD, NAD+/ NADH ratios must be well regulated by several complex systems to maintain redox homeostasis. In this context, we here provide evidence suggesting that tNOX also participates in modulating the cellular NAD+/NADH ratio in cancer cells. Thus, it is not surprising that tNOX also plays a role in capsaicin-induced apoptosis in this system. In fact, we previously discovered that capsaicin has divergent effects on the growth of gastric cancer cells that parallel its effects on tNOX expression, apoptosis in association with reduced tNOX expression in SNU-1 gastric cancer cells, and little apoptosis accompanied by insignificant changes in tNOX in the TMC-1 metastatic gastric carcinoma line.4 However, whether the intracellular NAD+/NADH ratio also contributed to the different cellular responses between these two gastric cancer lines was not examined. Experiments utilizing tNOX-knockdown cells showed that tNOX-depletion decreased the intracellular NAD+/NADH ratio compared to that in control cells (Figure 5), further confirming a regulatory role of tNOX in maintaining the NAD+/NADH redox status. Our results thus provide yet another scenario in which capsaicin regulates intracellular NAD+/NADH ratio and SIRT1 activity in A549 cancer cells, in this case through tNOX protein. In contrast, the lack of tNOX expression in the noncancer MRC-5 line resulted in an attenuated inhibitory effect of capsaicin on the intracellular NAD+/NADH ratio that acted concurrently with capsaicin-induced SIRT1 activity (measured in vitro) to ultimately deacetylate p53 and induce autophagy. Notwithstanding being generally considered a chemopreventive agent, capsaicin has demonstrated mutagenic properties53 and tumor-promoting effect.42,54−58 Moreover, data from epidemiologic studies suggest that capsaicin may exert dual effectsantitumor or tumor-promotingdepending

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AUTHOR INFORMATION

Corresponding Author

*(P.J.C.) Mail: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan. Phone: +886 4 22840896. Fax: +886 4 22853469. E-mail: pjchueh@ dragon.nchu.edu.tw. Present Address Δ

Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.

Author Contributions

Y.-H.L. and P.J.C. conceived and designed the experiments. Y.H.L. H.-Y.C., and L.J.S. performed the experiments. Y.-H.L. H.Y.C., L.J.S., and P.J.C. analyzed the data. Y.-H.L. and H.-Y.C contributed reagents/materials/analysis tools. Y.-H.L. and P.J.C. wrote the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Tamotsu Yoshimori at Osaka University in Japan for providing pEGFP-LC3 plasmid. REFERENCES

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