Effect of Allyl Sulfides from Garlic Essential Oil on Intracellular Ca2+

Nov 19, 2012 - Compound 3 also induced a [Ca2+]i elevation when extracellular Ca2+ was removed, ... Elevation of [Ca2+]i caused by 3 in the Ca2+-conta...
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Effect of Allyl Sulfides from Garlic Essential Oil on Intracellular Ca2+ Levels in Renal Tubular Cells Chung-Ren Jan,† Horng-Ren Lo,‡ Chung-Yi Chen,‡ and Soong-Yu Kuo*,‡ †

Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, Kaohsiung 83102, Taiwan

‡

ABSTRACT: Diallyl sulfide (1), diallyl disulfide (2), and diallyl trisulfide (3), which are major organosulfur compounds of garlic (Allium sativum), are recognized as a group of potential chemopreventive compounds. In this study, the early signaling effects of 3 were examined on Madin-Darby canine kidney (MDCK) cells loaded with the Ca2+-sensitive dye fura-2. It was found that 3 caused an immediate and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50 = 40 μM). Compound 3 also induced a [Ca2+]i elevation when extracellular Ca2+ was removed, but the magnitude was reduced by 45%. In Ca2+-free medium, the 3-induced [Ca2+]i level was abolished by depleting stored Ca2+ with 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Elevation of [Ca2+]i caused by 3 in the Ca2+-containing medium was not affected by modulation of protein kinase C activity. The 3-induced Ca2+ influx was inhibited by nifedipine and nicardipine (1 μM). U73122, an inhibitor of phospholipase C, abolished ATP (but not the 3-induced [Ca2+]i level). These findings suggest that 3 induced a significant [Ca2+]i elevation in MDCK renal tubular cells by stimulating both extracellular Ca2+ influx and thapsigargin-sensitive intracellular Ca2+ release via as yet unidentified mechanisms. Furthermore, the order of the allyl sulfide-induced [Ca2+]i elevation and cell viability was 1 < 2 < 3. The differential effect of allyl sulfides on Ca2+ signaling and cell death appears to correlate with the number of sulfur atoms in the structure of these allyl sulfides.

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everal population-based studies show that inhabitants of Southeast Asian countries have a much lower risk of colon, gastrointestinal, prostate, breast, and other cancers than their European and American counterparts.1 It is very likely that constituents of their diet such as garlic, ginger, and chillies may play important roles in cancer prevention. Garlic (Allium sativum L.; Alliaceae) has been used widely as a flavoring agent in cooking and as a medicinal herb in traditional medicine.2 Garlic-derived organosulfur compounds have been reported to reduce chemically induced colon, esophageal, lung, mammary, pulmonary, skin, and stomach tumors.3−7 Among these sulfurcontaining compounds, diallyl sulfide (1), diallyl disulfide (2), and diallyl trisulfide (3) are the three major components in garlic volatile oil. The potential chemopreventive effects of allyl sulfides have been attributed not only to the modulation of the antioxidative and/or drug-metabolizing enzyme systems, but also to the inhibition of cell proliferation and induction of apoptosis for tumor cells.8 Various pathways of these essential garlic-derived allyl sulfides have been suggested for anticarcinogenic activity, including modulation of xenobiotic-metabolizing enzyme activities,9 inhibition of DNA adduct formation,10 modulation of signal transduction pathways,11 reduction of proliferation,12 and induction of apoptosis.12 Recently, molecular changes of allyl sulfides in G2/M arrest,12,13 β-tubulin oxidation,14 and increase of cytosolic Ca2+ ([Ca2+]i) were shown.15,16 In addition, the possible role of Ca2+ in apoptosis induced by diallyl disulfide (2) was described in colon cancer cells and retinal ganglion cells.17,18 It has been shown that BAPTA (an intracellular Ca2+ chelator) can suppress 2-evoked [Ca2+]i © 2012 American Chemical Society and American Society of Pharmacognosy

elevation, and that reactive oxygen species (ROS) generation can prevent caspase 3 activation and apoptosis. However, despite the accumulation of data, the underlying molecular mechanism of Ca2+ signaling by diallyl trisulfide (3) is still unclear. It is known that Ca2+ ions serve as a ubiquitous second messenger in all eukaryotic cells.19 Under physiological conditions, the resting [Ca2+]i is maintained at levels less than 0.1 μM, about four orders of magnitude lower than in the extracellular environment (1−2 mM), but cellular excitation induces a transient [Ca2+]i elevation up to several mM, or to even higher levels in tiny cellular compartments. These transient fluctuations of [Ca2+]i (termed the “Ca2+ signal”) trigger or regulate various intracellular events. It is well established that cellular Ca2+ overload, or perturbation of intracellular [Ca2+]i levels, may cause cytotoxicity and result in either apoptosis, necrosis, or autophagy. In general, the generation of Ca2+ signal is determined by interaction of (1) external Ca2+ entry, (2) Ca2+ release from intracellular compartments (Ca2+ stores), (3) cytoplasmic Ca2+ buffering by Ca2+ binding proteins, and (4) subsequent Ca2+ removal from the cytoplasm due to transmembrane Ca2+ efflux or sequestration by intracellular Ca2+ stores located in the organelles.20 The aim of the present study was to explore the effect of the allyl sulfides 1-3 on [Ca2+]i in renal tubular cells. Madin-Darby Received: July 30, 2012 Published: November 19, 2012 2101

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canine kidney (MDCK) cells have properties akin to human renal tubular cells and have been used successfully as a model for kidney research. Many endogenous and exogenous agents can stimulate MDCK cells by causing a [Ca2+]i rise, such as ATP,21 bradykinin,22 methylglyoxal,23 and [6]-gingerol.24 The inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store is important in releasing Ca2+ into the cytosol when cells are stimulated by endogenous agents such as ATP and bradykinin;21,22 but exogenous agents can release Ca2+ in an IP3-independent manner.24 The Ca2+ release may induce Ca2+ influx across the plasma membrane via the process of storeoperated Ca2+ entry or other undefined pathways.25 Using fura-2 as a fluorescent Ca2+ indicator, this study shows that diallyl trisulfide (3) induced a significant and prolonged [Ca2+]i rise in MDCK cells. The time course and the concentration-response relationship, the Ca2+ sources of the Ca2+ signal, and the role of phospholipase C in the signal, have been explored. The effects of allyl sulfides on cell viability and on the cell cycle have also been examined.

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RESULTS AND DISCUSSION

Diallyl trisulfide (3) at concentrations between 25 and 100 μM increased [Ca2+]i in a concentration-dependent manner in the presence of extracellular Ca2+. Figure 1A shows typical recordings of the [Ca2+]i elevation induced by 25−100 μM 3. At a concentration of 1 μM, 3 had no effect on [Ca2+]i (i.e., equivalent to baseline, 0 μM). The [Ca2+]i rise induced by 25− 100 μM 3 comprised an immediate rise and a sustained phase within 250 s. At a concentration of 100 μM, the [Ca2+]i rise had a net value of 145 ± 2 nM at 250 s. Figure 1D (filled circles) shows the concentration−response curve of 3-induced responses. Figure 1B shows that 100 μM 3 induced an increase in the 340 nm excitation signal accompanied by a corresponding decrease in the 380 nm excitation signal. This suggests that the rises in fura-2 340/380 ratio signals induced by 3 reported changes in [Ca2+]i instead of artifacts. Experiments were performed to evaluate the relative contribution of extracellular Ca2+ entry and stored Ca2+ release in the response to 3. Figure 1C shows that removal of extracellular Ca2+ partly suppressed the 3-induced [Ca2+]i rise. The concentration-response relationship of 3-induced [Ca2+]i elevation in the presence and absence of extracellular Ca2+ is

Figure 1. Effects of diallyl trisulfide (3) on [Ca2+]i in MDCK cells. (A) Concentration-dependent effects of 3. The concentration of the reagent is indicated. The experiments were performed in Ca2+-containing medium. Compound 3 was added at 30 s and was present throughout the measurement of 250 s. (B) Changes induced by 3 in the 340 and 380 nm excitation wavelength signals (emission wavelength 510 nm). Compound 3 (100 μM) was added at 30 s. (C) Effect of extracellular Ca2+ removal on 3-induced [Ca2+]i rise. The concentration of 3 is indicated. (D) Concentration−response plots of 3-induced [Ca2+]i rises in Ca2+-containing medium (filled circles) and Ca2+-free medium (open circles). The data are presented as the percentage of control, which is the net [Ca2+]i rise induced by 100 μM 3 in Ca2+-containing medium. Data are means ± SEM of five experiments (*p < 0.05 compared to open circles). 2102

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Figure 2. Intracellular sources of 3-induced [Ca2+]i elevation. All experiments were performed in Ca2+-free medium. Compounds were applied at the time indicated by the arrows. The concentration was 100 μM for 3 and 1 μM for thapsigargin. Data are means ± SEM of five experiments.

Figure 3. Effect of U73122 on 3-induced [Ca2+]i elevation. (A) U73122 (2 μM), ATP (10 μM), and 3 (100 μM) were added at time points indicated. (B) ATP (10 μM) was added at 30 s. All experiments were performed in Ca2+-free medium. Data are means ± SEM of five experiments.

Figure 4. Effect of Ca2+ blockers on 3-induced [Ca2+]i elevation. All experiments were performed in Ca2+-containing medium. (A) Trace a: 3 (100 μM) was added at 30 s. Trace b and trace c: Nifedipine or nicardipine (1 μM) was added to cells 1 min before 3. (B) The data are presented as the percentage of control, which is the net area under the curve (30−250 s) of the [Ca2+]i rise induced by 100 μM 3 (trace a in A). Data are means ± SEM of five experiments (*p < 0.05 compared to control).

itor,28 caused a [Ca2+]i rise that comprised an initial increase and a gradual decay toward baseline. The net maximum [Ca2+]i value was 136 ± 4 nM (n = 5). After depleting the endoplasmic reticulum Ca2+ store with thapsigargin, addition of 100 μM 3 did not induce a [Ca2+]i elevation, as shown in Figure 2A. Conversely, Figure 2B shows that after preincubation with 3 (100 μM) for 220 s, addition of 1 μM thapsigargin did not induce any [Ca2+]i elevation.

shown in Figure 1D. Ca2+ removal inhibited the [Ca2+]i rise caused by 100 μM 3 by 45% in terms of the maximum value (n = 5; p < 0.05). The role of the endoplasmic reticulum Ca2+ stores in the 3induced [Ca2+]i elevation was examined because previous studies show that these stores play a key role in Ca2+ release in MDCK cells.27 Figure 2A shows that application of 1 μM thapsigargin, an endoplasmic reticulum Ca2+-ATPase inhib2103

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Figure 5. Effect of 1−3 on [Ca2+]i. (A) Each allyl sulfide (50 μM) was added at 30 s and was present throughout the measurement of 250 s. Traces a, b, and c represent diallyl sulfide (1), diallyl disulfide (2), and diallyl trisulfide (3), respectively. The experiments were performed in Ca2+-containing medium. (B) The y axis is the percentage of the control response that was the net area under the curve (30−250 s) of the [Ca2+]i rise induced by 3 (trace c in A). Results are expressed as the percentage of control, which is the response induced by 3. Data are means ± SEM of five experiments (*p < 0.05 compared with control).

Previous studies have shown that in MDCK cells, stored Ca2+ can be released by pathways dependent on or independent of phospholipase C activity.29,24 Thus, an effort was made to explore whether phospholipase C plays a role in 3-induced Ca2+ release. Figure 3A shows that in Ca2+-free medium, addition of 2 μM U73122 to suppress phospholipase C activity did not alter the basal [Ca2+]i but did abolish the [Ca2+]i rise induced by ATP (10 μM) (n = 5), a phospholipase C-dependent Ca2+ mobilizer.30,21 The control ATP (10 μM)-induced [Ca2+]i had a net peak value of 132 ± 3 nM (n = 5) (Figure 3B). Conversely, U73343 (10 μM), an inactive U73122 analogue, did not affect ATP-induced [Ca2+]i rise (not shown).30 This suggests that U73122 effectively suppressed phospholipase C activity. Figure 3A further shows that 100 μM 3 added after ATP induced a [Ca2+]i rise that was similar to the control response shown in Figure 1C (n = 5). To explore the pathways underlying 3-induced Ca2+ entry, the effects of different L-type Ca2+ entry blockers on 3-induced [Ca2+]i rise were explored. Figure 4A shows that in Ca2+containing medium, pretreatment with 1 μM nicardipine or 1 μM nifedipine inhibited 100 μM 3-induced [Ca2+]i rise by 78.8% and 42.2%, respectively (n = 5; p < 0.05). However, the Ca2+ influx was not affected by 1 μM diltiazem and 1 μM verapamil (n = 5, Figure 4B). The role of protein kinase C in 3-induced [Ca2+]i rise was investigated. It was found that 100 μM 3-induced [Ca2+]i rise was not altered by pretreatment with 10 nM phorbol myristate acetate (a protein kinase C activator) or 2 μM GF109203 X (a protein kinase C inhibitor) (n = 5; data not shown). To explore the role of the number of sulfur atoms in allyl sulfide-induced [Ca 2+] i rises, the effects of equimolar concentrations of allyl sulfides 1-3 on [Ca2+]i were examined. Figure 5A shows that in Ca2+-containing medium, [Ca2+]i was not significantly increased when cells were treated with 50 μM diallyl sulfide (1). Diallyl disulfide (2)-induced [Ca2+]i rises were 45.6 ± 1.2% of diallyl trisulfide (3)-induced responses (n = 5; p < 0.05). Figure 5B compares the net (baseline subtracted) maximum value of the three allyl sulfide-induced [Ca2+]i rises. The order of the magnitude of the [Ca2+]i rises caused by the three allyl sulfides was: 1 < 2 < 3. Since oxidative stress-induced sustained [Ca2+]i rises often lead to cytotoxicity,31 experiments were performed to explore

the effect of the three organosulfur compounds on the viability of MDCK cells using the WST-1 assay. Figure 6 shows that

Figure 6. Cytotoxic effect of allyl sulfides on viability of MDCK cells. The cell viability assay is described in the Experimental Section. Different concentrations of allyl sulfides were added to cells for 16 h. Data are expressed as the percentage of the control (allyl sulfides were absent). The control had 11 812 ± 687 cells/well before experiments and 16 632 ± 723 cells/well after incubation for 16 h.

treatment with these allyl sulfides (0−2 mM, except 3) for 16 h decreased cell viability in a concentration-dependent manner (n = 5; p < 0.05). The IC50 values of diallyl sulfide (1), diallyl disulfide (2), and diallyl trisulfide (3) were 3.26, 1.82, and 0.05 mM, respectively. Figure 7 depicts representative histograms for cell-cycle distribution in cells following 16 h exposure to dimethyl sulfoxide (DMSO), and 100 μM 1-3, respectively. The flow cytometric data demonstrated that approximately 33.1% of the cells showed apoptotic DNA in cells treated with 100 μM 3. The percentage of the G2/M fraction was increased about 2-fold in the 100 μM 3-treated group, compared with the DMSO-treated control. Conversely, only about 7% of apoptotic fraction was detected in 100 μM 2-treated cells under the same conditions. These results demonstrated that 3-induced cell damage included apoptosis and G2/M phase arrest. The data suggest that the order of the magnitude of apoptosis caused by these organosulfur compounds in MDCK cells was: 1 < 2 < 3. 2104

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Figure 7. Effect of diallyl sulfide (1), diallyl disulfide (2), and diallyl trisulfide (3) on the cell cycle of MDCK cells. Cells were exposed to DMSO and 100 μM 1−3 for 16 h. After exposure, cells were harvested and fixed in DMEM−methanol (1:2; v/v) solution, stained with propidium iodide, and subjected to flow cytometric analysis. Data represented in each panel are the percentages of SubG1, G1, S, and G2/M in the cell cycle. These experiments were performed at least three times, and a representative experiment is presented.

On removal of extracellular Ca2+, the 3-induced [Ca2+]i elevation displayed a smaller [Ca2+]i elevation throughout the 250-s measurement. This suggests that the Ca2+ influx contributed not only to the initial increase, but also to the prolonged phase of 3-induced [Ca2+]i signaling in the Ca2+containing medium. In non-excitable cells, a possible Ca2+ influx pathway is store-operated Ca2+ entry, a process initiated by depletion of Ca2+ stores.25 This possibility was not explored due to the lack of selective pharmacological inhibitors for this Ca2+ influx.41 Thus, it remains possible that Ca2+ entry mechanisms other than depletion-activated channels may be crucial in Ca2+ influx in non-excitable cells. The extent of [Ca2+]i elevation, cell viability, and sub-G1 portions were correlated to the amount of sulfur number and were in the order of diallyl trisulfide (3) > diallyl disulfide (2) > diallyl sulfide (1). Recent studies have revealed that the number of sulfur elements may play a critical role in the biological activities of garlic-derived organosulfur compounds.41,12,34 The allyl sulfides that contain two sulfur atoms are more potent in inhibiting benzo[a]pyrene-induced forestomach neoplasia than 1 (containing only one sulfur atom).42 Meanwhile, the antiproliferative effect of 3 on human skin cells was related to the highly reactive sulfane sulfur groups.16 Reactive disulfide compounds are a group of oxidizing agents that can produce ROS, which are thought to oxidize the cell membrane, leading to cell injury and death during inflammation, aging, radiation, and ischemia-reperfusion of the heart, kidney, liver, intestines, and brain.43−45 Cells exposed to some thiol agents are recognized to change the cellular redox state and key enzymes involved in cell function and growth. Disruption of this homeostasis is mostly followed by dysregulation in [Ca2+]i and cell growth.46,47 Therefore, the antiproliferative effects of 3 on cells may relate to modulation of thiols in the cytoplasm and cellular membrane. Collectively, this study shows that in MDCK cells, diallyl trisulfide (3) caused [Ca2+]i elevation in a concentrationdependent manner by evoking phospholipase C-independent Ca2+ release from the endoplasmic reticulum and also by causing Ca2+ influx via a nifedipine-sensitive pathway. Compound 3 also induced cytotoxicity via apoptosis. These effects may play a crucial role in the physiological action of 3.

Diallyl trisulfide (3) induces growth arrest in the G2/M phase and cytotoxicity in human liver tumor cells and prostate cancer cells.12,13,32 This compound also causes [Ca2+]i rises in human A549 lung tumor cells and skin cancer cells.15,16 However, the source of the increased intracellular Ca2+ of 3 is still unexplored. The present results suggest that 3 caused a significant concentration-dependent, sustained [Ca2+]i rise in MDCK cells. In Ca2+-containing medium, the [Ca2+]i level induced by 3 was sustained without a decay during the 5 min of measurements. Sustained [Ca2+]i elevation is thought to alter many cell functions.31 Garlic-derived organosulfur compounds may affect cell physiology significantly by changing Ca2+ signaling and stimulating Ca2+-coupled bioactive molecules. The results show that the [Ca2+]i elevation was contributed by both intracellular Ca2+ release and extracellular Ca2+ influx, because the signal was partly suppressed by removal of extracellular Ca2+. Compound 3 and thapsigargin appear to share a common endoplasmic reticulum Ca2+ store, because the response to 3 was mostly inhibited by depletion of the endoplasmic reticulum Ca2+ store with thapsigargin, and conversely, thapsigargin failed to release additional Ca2+ after treatment with 3. Similar observations were also examined as a result of manipulation by [6]-gingerol and diallyl sulfide (1) in MDCK cells.24,33 The endoplasmic reticulum is one of the major Ca2+ stores where various proteins and lipids are synthesized and modified.34,35 Perturbation of endoplasmic reticulum Ca2+ homeostasis, protein misfolding, or oxidative stress can lead to cell death.35,36 How 3 releases Ca2+ store from the endoplasmic reticulum is unclear, but the process seems to be independent of phospholipase C activity because suppression of this protein did not affect 3-induced Ca2+ release. Since 3 and thapsigargin share the same Ca2+ stores, 3 may very likely release Ca2+ in a manner similar to thapsigargin by inhibiting the endoplasmic reticulum Ca2+ pump. Ryanodine receptor-dependent Ca2+ stores are not detected in MDCK cells.22 The diallyl trisulfide (3)-induced Ca2+ influx appears to be via a pathway sensitive to nicardipne and nifedipine. In MDCK cells, nifedipine-sensitive Ca2+-channels have been implicated in several reports. Nifedipine is suggested to inhibit Ca2+ influx induced by hyperosmolarity,37 alkaline-stress-induced membrane potential changes,38 and effects on 2-O-methyl PAF.39 This nifedipine-sensitive 3-induced Ca2+ influx was not via conventional L-type Ca2+ channels since it was not inhibited by diltiazem or verapamil. This is consistent with a previous study showing that MDCK cells are non-excitable.40

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EXPERIMENTAL SECTION

Cell Line, Chemicals, and Reagents. MDCK cells were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium. The medium was supplemented 2105

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Statistics. Data are reported as means ± SEM of five experiments. Data were analyzed by two-way analysis of variance (ANOVA) using the Statistical Analysis System (SAS, SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were performed by post-hoc analysis using the Tukey’s HSD (honestly significant difference) procedure. A p value of less than 0.05 was considered significant.

with 10% heat-inactivated fetal calf serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were kept at 37 °C in 5% CO2containing humidified air. The reagents for cell culture were from Gibco (Gaithersburg, MD, USA). Fura-2/AM was from Molecular Probes (Eugene, OR, USA). U73122 (1-[6-[[(17β)-3-methoxyestra1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) and U73343 (1-[6-[[17β]-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl)-2,5-pyrrolidinedione) were from Biomol (Plymouth Meeting, PA, USA). Diallyl sulfide (1; 97% purity by HPLC), diallyl disulfide (2; ≥80% purity), and diallyl trisulfide (3; ≥98% purity by HPLC) were purchased from Fluka Chemical Co. (Buchs, Switzerland), Tokyo Kasei Chemical Co. (Tokyo, Japan), and LKT Laboratories, Inc. (St. Paul, MN, USA), respectively. The purity of 2 was further purified by HPLC to ≥95%. These allyl sulfides were dissolved in dimethyl sulfoxide and stored at −20 °C. DMSO was used as a solvent control and was added at a dilution equivalent to the highest concentration of 3 tested in each assay. WST-1 was from Roche Molecular Biochemical (Indianapolis, IN, USA). The other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). Measurements of [Ca2+]i. Trypsinized cells (106/mL) were allowed to recover in the culture medium for 1 h before being loaded with 2 μM fura-2/AM for 30 min at 25 °C in the same medium. The cells were washed once with serum-free DMEM medium and resuspended in Ca2+-containing medium (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM D-glucose, and 2 mM HEPES, pH 7.4). Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25 °C) with continuous stirring; the cuvette contained 1 mL of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer (Kyoto, Japan) by recording excitation signals at 340 and 380 nm and an emission signal at 510 nm at 1 s intervals. Maximum and minimum fluorescence values were obtained by adding 0.1% Triton X-100 and 10 mM EGTA sequentially at the end of each experiment. [Ca2+]i was calculated as described previously assuming a Kd of 155 nM.26 In experiments that were performed in the absence of extracellular Ca2+, cells were bathed in Ca2+-free medium in which CaCl2 (2 mM) was substituted with 0.1 mM EGTA. Cell Viability Assays. Viability assays were carried out as previously described.23 The assay was based on cleavage of the tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5tetrazolio]-1,3-benzene disulfonate) by active mitochondria to produce a soluble colored formazan salt. Since this cleavage can be performed only in active cells, its magnitude was correlated directly with cell number. MDCK cells were plated at a density of 1 × l04 in 96-well microtiter plates (Corning Costar Italia, Milan, Italy). Twenty-four hours later, at 70% confluence, the growth medium was removed and replaced with the test solutions (100 μL). After a 16-h exposure, the medium was aspirated and the cells were washed twice with DMEM. Then, 100 μL of DMEM plus 10 μL of WST-1 were added to each well. The cells were incubated for 2 h at 37 °C in a humidified atmosphere with 5% CO2; then the microplate was thoroughly shaken for 1 min, and the absorbance was measured at 450 nm using a microtiter reader (model MRX II, Dynex Technologies, Chantily, VA, USA). Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and was expressed as a percentage of the control value, which was treated with vehicle only (0.1% DMSO), taken as 100% growth. Cell Cycle Analysis. Adherent and floating cells were pooled, washed with DMEM, then fixed in DMEM-methanol (1:2, v/v) solution and maintained at 4 °C for over 18 h as described previously.23 Following two more washes with DMEM, the cell pellet was stained with the fluorescent probe solution containing DMEM, 40 μg of propidium iodide/mL, and 40 μg of DNase-free RNaseA/mL for 30 min at room temperature in the dark. Cells were then analyzed using a FACS-Calibur cytometer (Becton Dickinson, San Jose, CA, USA) with excitation at 488 nm and collection of fluorescence emission above 620 nm. Doublets and clumps were gated out by pulse processing. The percentage of cells undergoing DNA damage was obtained from the percentage of cells in the distinct subdiploid region of the DNA distribution histograms.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +886-7-7811151-6200. Fax: +886-7-7863667. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from Veterans General Hospital-Kaohsiung (VGHKS95-037) to C.R.J. The authors thank Dr. C.-H. Chen (National Chiayi University) for the flow cytometric analyses.

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REFERENCES

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dx.doi.org/10.1021/np3005248 | J. Nat. Prod. 2012, 75, 2101−2107