ANTICANCER RESEARCH 34: 1829-1836 (2014)

Induction of Apoptosis by Deinoxanthin in Human Cancer Cells

YONG-JI CHOI1, JUNG-MU HUR1, SANGYONG LIM1, MINHO JO1, DONG HO KIM1 and JONG-IL CHOI2

1Research Division for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup, Republic of Korea; 2Department of Biotechnology and Bioengineering, Chonnam National University, Gwangju, Republic of Korea

Abstract. Background: Deinoxanthin is unique carotenoid activity and their potential for preventing epithelial cancer and isolated from the radioresistant bacterium Deinococcus chronic diseases (2, 3). Carotenoids are found in many radiodurans. In the present study, the induction of apoptosis bacteria and occur in a wide range of phyla in both of cancer cells by deinoxanthin was investigated. Materials photosynthetic and non-photosynthetic species. Deinococcus and Methods: Apoptotic effects were evaluated in HepG2, radiodurans is a red-pigmented, non-photosynthetic species PC-3, and HT-29 cells, and were measured through cell of bacteria well-their known for high resistance to γ-ray, viability, morphological changes, and a DNA fragmentation ultraviolet radiation, oxidants, and desiccation (4). Most assay. Intracellular generation of reactive oxygen species damaging effects of ionizing radiation on biological (ROS) was measured using 5-(and 6-)-carboxy-2’,7’- macromolecules are due to reactive oxygen species (ROS) dichlorodihydrofluorescein diacetate (carboxy-H2DCF-DA). produced by water radiolysis (5). The ROS-scavenging The expression of apoptotic and anti-apoptotic proteins was system in D. radiodurans consists of antioxidant enzymes, assayed by western blotting. Results: The half-maximal such as superoxide dismutase and catalase, and non- inhibitory concentration (IC50) values for deinoxanthin enzymatic antioxidants including Mn(II), pyrroloquinoline- against the HepG2, HT-29, and PC-3 cell lines were 59 μM, quinone, and carotenoids (6). 61 μM, and 77 μM, respectively. Deinoxanthin treatment Among the non-enzymatic antioxidants of D. radiodurans, caused an increase in ROS in all tested cells, suggesting carotenoids are efficient scavengers of ROS, particularly of possible pro-oxidant activity of deinoxanthin. Pro-caspase- singlet oxygen. Deinoxanthin, a unique carotenoid 3 was degraded in cancer cells by deinoxanthin treatment. synthesized by D. radiodurans that gives the bacterium its Moreover, BCL2 expression decreased, but that of BAX characteristic red color, has been identified as (all-E)-2,1’- increased. Conclusion: The present findings demonstrate dihydroxy-3’-4’-didehydro-1’,2’-dihydro-β,ψ-carotene-4-one for the first time the novel functional property of (7). Deinoxanthin is a stronger ROS scavenger than both deinoxanthin isolated from radioresistant bacteria as a carotenes (lycopene and β-carotene) and xanthophylls potent inducer of apoptosis in cancer cells. These data (zeaxanthin and lutein) (6, 8). The higher antioxidant activity suggest that deinoxanthin could be potentially useful as a of deinoxanthin may be ascribed to its chemical structure, chemopreventive agent. which is different from those of other carotenoids. Some carotenoids, such as lutein, lycopene, β-carotene, Carotenoids are one of the most common natural pigments, and fucoxanthin, are highly effective at suppressing and more than 600 different carotenoids can be found in carcinogenesis in animals and can induce apoptosis of nature (1). Although some natural carotenoids such as various cancer cell lines (9). Moreover, epidemiological astaxanthin, lycopene, and β-carotene have been used as food studies suggest that ingesting carotenoids reduces the risk of colorants for many years, it is only recently that they have prostate cancer. Deinoxanthin protects against DNA damage, been the subject of intense study with respect to antioxidant as it is a more efficient H2O2 and singlet oxygen scavenger than lutein, lycopene, or β-carotene (6, 8). Therefore, it would be interesting to investigate the effect of deinoxantin on cancer cells. Correspondence to: Jong-il Choi, Department of Biotechnology and However, no investigations concerning the anticancer Bioengineering, Chonnam National University, Gwangju 500-757, activity of deinoxanthin have been conducted as far as we South Korea. Tel: +82 625301846, Fax: +82 625301949, e-mail: are aware of. Therefore, in this study, the anticancer effects [email protected] of deinoxanthin isolated from D. radiodurans were assessed. Key Words: Deinoxanthin, apoptosis, cancer cells, reactive oxygen Additionally, the pathway leading to cancer cell death was species. extensively studied.

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Materials and Methods

Bacterial strains and growth conditions. D. radiodurans ATCC 13939 (American Type Culture Collection, Manassas, VA, USA) was grown at 30˚C in TGY (0.5% tryptone, 0.3% yeast extract, and 0.1% glucose) broth or on TGY agar (with 1.5% agar). Cell density in liquid culture was determined at 600 nm using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Deinoxanthin preparation. Carotenoid from D. radiodurans was extracted as follows. Exponential-phase cells were harvested by centrifugation at 5000 ×g for 10 min. The average pellet weight was 2.5 g/l culture. After three washes with sterilized water, the cell pellet Figure 1. The effect of deinoxanthin on viability of HepG2, HT-29, and was extracted three times with cold methanol in the dark. The PC-3 cells. Values are the means±standard errors of three different methanolic extracts were combined and concentrated under rotary experiments. Values with different letters (a-f) differ significantly among vacuum (Eyela, Tokyo, Japan). The residue was dissolved in 100 ml of the concentration (p<0.05). Values with different letters (x-z) differ ethyl acetate and shaken with deionized water to remove salts. The significantly regarding concentration (p<0.05). organic layer was dried over magnesium sulfate and concentrated under reduced pressure. Deinoxanthin in the extracts was analyzed by high-performance liquid chromatography using a Waters 2690 Alliance system (Milford, MA, USA) following the method of Tian et al. (6). maximum of 460 nm. After treating HepG2 cells with 60 μM, HT- 29 cells with 60 μM, and PC-3 cells with 80 μM, respectively, Human cancer cell culture. Human hepatoma HepG2 and prostate deinoxanthin for 18 and 24 h, morphological changes, including PC-3 cells were obtained from the ATCC and human colon cancer volume and nuclear chromatin condensation were recorded. The HT-29 cells were obtained from Korean Cell Line Bank (Seoul, morphology of human cancer cells exposed to deinoxanthin for South Korea). Cells were maintained in RPMI-1640 medium different times was observed under an inverted microscope. Hoechst supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 33258 staining was applied to observe apoptotic morphology. The and 50 mg/ml streptomycin at 37˚C in a 5% CO2 incubator (Sanyo, cells were fixed in 10% formaldehyde in phosphate-buffered saline Osaka, Japan). Cell culture reagents were obtained from Gibco BRL (PBS) for 10 min, stained with Hoechst 33528 (10 mg/ml) for 1 h, (Carlsbad, CA, USA). and then subjected to fluorescence microscopy (Eclipse TE2000-E; Nikon, Tokyo, Japan). Determination of cell viability. The 3-(4,5-dimethylthiazil-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was performed to ROS measurement. HepG2, HT-29, and PC-3 cells were seeded in evaluate deinoxanthin cytotoxicity. Cells were seeded in 24-well six-well plates at 2×104 cells/well and allowed to attach overnight. plates at a density of 4×104 cells/well and treated with deinoxanthin The cells were treated with or without deinoxanthin and incubated dissolved in dimethyl sulfoxide for 24 h. After the treatment, the with 5 mM 5-(and 6-)-carboxy-2’,7’-dichlorodihydrofluorescein medium was removed, and the cells were washed with phosphate- diacetate (carboxy-H2DCF-DA; Invitrogen, Carlsbad, CA, USA) at buffered saline (PBS). Fresh medium was added, and the cells were 37˚C for 15 min. The cells were washed twice with PBS, trypsinized, incubated with 100 ml of 1 mg/ml MTT for 3 h. The number of and resuspended in OptiMem I medium (Invitrogen). The rate of viable cells was directly proportional to the production of formazan, oxidation of the dye in the cells was monitored by measuring which was solubilized in isopropanol and measured fluorescence with a flow cytometer (Beckman), using an excitation spectrophotometrically at 570 nm. Wells without deinoxanthin or wavelength of 480 nm and an emission wavelength of 530 nm. cells were used as blanks. Protein extraction and western blot analysis. Cells were seeded at Apoptosis assay using flow cytometry. Cells were seeded onto six-well 2×106 cells per dish containing 10 ml of medium in 10 cm dishes plates at 2×104 cells/well and pretreated with deinoxanthin (60 μM and treated with deinoxanthin as described above for the MTT assay. for HepG2, 60 μM for HT-29, and 80 μM for PC-3) for 24 h. The After deinoxanthin treatment, the medium was removed, and the cells were then trypsinized and gently washed with serum-containing cells were rinsed twice with PBS. After adding 0.6 ml of cold RIPA culture medium, followed by PBS. The cells were resuspended in buffer [10 mM Tris(pH 7.5), 0.1% SDS, and 1% Triton ×100) and binding buffer (10 mM HEPES, 140 mM NaCl, and 25 mM CaCl2) protease inhibitors cocktail (Sigma-Aldrich, St. Louis, MO, USA), and incubated with annexin V-FITC and propidium iodide (PI; MBL the cells were scraped at 4˚C. The cell lysate was then centrifuged International, Nagoya, Japan) at room temperature for 15 min. The at 10,000 ×g at 4˚C for 10 min. The proteins obtained were separated supernatant was removed, and the cells were stained with annexin V- by electrophoresis on a 12% polyacrylamide gel and transferred to a FITC and PI according to the manufacturer’s instructions. Untreated nitrocellulose membrane. The membrane was stained with Ponceau- cells were used as a control for double staining. Fluorescence analysis S to confirm the transfer of all samples and then incubated in a was carried out using a flow cytometer (Beckman, Miami, FL, USA). blocking solution of PBS with 0.05% Tween 20 (PBST) and 5% non- At least 50,000 cells were counted in each measurement. fat powered milk at room temperature for 1 h. The membranes were reacted with the following antibodies: rabbit polyclonal antibody to Hoechst 33258 staining. Hoechst 33258 is excited by ultraviolet BAX (Sigma-Aldrich), rabbit polyclonal antibody to BCL2 (Cell light at 350 nm and emits blue/cyan fluorescent light at an emission Signaling Technology, Danvers, MA, USA), rabbit polyclonal

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Figure 2. Induction of apoptosis by deinoxanthin in human cancer cells. HepG2 (A), HT-29 (B), and PC-3 (C) cells were treated with 60, 60, and 80 μM deinoxanthin for 24 h. Lower right quadrant, early-apoptotic cells, i.e., annexin V-FITC-positive/propidium iodide (PI)-negative cells; upper right quadrant, late-apoptotic cells, i.e., annexin V-FITC-positive/PI-positive cells.

antibody to caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA, PC-3 cells were 59 μM, 61 μM, and 77 μM, respectively. USA), and rabbit monoclonal antibody to β-actin (Cell Signaling These data indicate that a 24-h deinoxanthin treatment Technology) at a dilution of 1:1000 for 90 min. Extensive washes induced a concentration-dependent decrease in total cell were then performed with PBST. The membranes were then viability. Therefore, deinoxanthin was an effective cytotoxic incubated with 1:1000 horseradish peroxidase-conjugated secondary antibodies (Zymed, San Francisco, CA, USA) for 1 h, washed with agent against the human cancer cells tested in this study. PBST, and developed using the enhanced chemiluminescence detection system (Amersham Pharmacia, Buckinghamshire, UK). Induction of apoptosis by deinoxanthin. After a 24-h treatment with deinoxanthin, the cells were stained with Statistical analysis. The statistical analysis was performed by one- annexin V/PI to confirm induction of apoptosis. The way analysis of variance, and the differences among mean values percentage of apoptotic cells (early and late) induced by were identified by Duncan’s multiple range test using SAS release deinoxanthin at the IC value is shown in Figure 2. 8.01 software (SAS Institute Inc., Cary, NC, USA). A value of 50 p<0.05 was considered significant. Mean values and standard errors Deinoxanthin induced a higher percentage of apoptotic cells are reported. in HepG2 than HT-29 and PC-3 cells. Apoptosis measurements in HT-29 and PC-3 were similar. These results Results indicate that deinoxanthin significantly induced apoptosis in the three human cancer cell lines. Effects of deinoxanthin on growth of human cancer cells. The Apoptotic bodies can also be observed with a inhibitory activity of deinoxanthin against growth of human fluorescence microscope after staining with Hoechst cancer cells were investigated using the MTT assay. Figure 1 33258. HepG2, HT-29, and PC-3 cells were stained with shows the growth-inhibitory effects of deinoxanthin against Hoechst 33258 dye after exposure to deinoxanthin for 18 the three cancer cell lines at different concentrations (20, 40, and 24 h. The dye stains the condensed chromatin of 80, and 100 μM). Inhibition of the three cell lines by apoptotic cells more brightly than the chromatin of normal deinoxanthin was similar but was most effective in HepG2 cells (Figure 3). After the cells were treated with cells. Cell viability decreased depending on the deinoxanthin deinoxanthin, marked morphological changes due to cell concentration used. At 100 μM, cell viabilities were 12.1% apoptosis, such as chromatin condensation and nuclear for HepG2, 17.27% for HT-29, and 22.4% for PC-3, fragmentation, were observed. The number of apoptotic respectively. The half-maximal inhibitory concentration cells increased gradually in a time-dependent manner in (IC50) values for deinoxanthin against HepG2, HT-29, and the human cancer cell lines.

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Figure 3. Apoptosis observed by Hoechst 33258 staining (×100) after deinoxanthin treatment. Apoptosis of the three cancer cell lines was observed after deinoxanthin treatment for 18 and 24 h. The marked morphological changes due to cell apoptosis, such as chromatin condensation and nuclear fragmentation, were clearly shown using Hoechst 33258 staining.

ROS in human cancer cells. Cells are committed to apoptosis three human cancer cells treated with deinoxanthin. As after loss of outer mitochondrial membrane integrity and the shown in Figure 5, pro-caspase-3 was reduced in cancer cells release of cytochrome c from the mitochondria to the by deinoxanthin. Moreover, BCL2 expression level cytosol. The production of ROS contributes to mitochondrial decreased, whereas that of BAX increased in the tested damage that may facilitate further release of ROS into the cancer cell lines following deinoxanthin treatment. These cytoplasm (10). To explore the role of ROS in the results demonstrate that deinoxanthin significantly induced mechanism of action of deinoxanthin, we examined whether the release of BAX. In addition, caspase-3 appears to be deinoxanthin could increase the level of intracellular ROS in activated by deinoxanthin treatment. the three cancer cell lines using DCF fluorescence. Intracellular generation of ROS was measured using Discussion carboxy-H2DCF-DA, which is a cell-permeable dye. This compound is oxidized inside cells by ROS to form Several reports have shown the induction of apoptosis by fluorescent carboxydichlorofluorescein. HepG2, HT-29, and carotenoids from plants, dinoflagellates and algae (11-14), RC-3 cells were treated with deinoxanthin at concentrations but none from radioresistant bacteria. Therefore, this study of 60, 60, and 80 μM, respectively, for 24 h, and then was carried out to determine whether deinoxanthin affected analyzed for ROS using flow cytometry (Figure 4). The the viability of human cancer cells and apoptosis. relative ROS value increased to 48.4% in HepG2, 17.9% in We used three different cancer cell lines: HepG2, which HT-20, and 47.6% in PC-3 cells following deinoxanthin modeled polarized human hepatocytes; HT-29, a human treatment. These data indicate that the increase in colon cancer cell line; and PC-3, human prostate cancer intracellular ROS might play a role as an early mediator of cells, because these types of cancers consist a serious deinoxanthin-induced apoptosis. problem in most developed countries (15). Deinoxanthin inhibited cell growth in a dose-dependent manner. Among Effect of deinoxanthin on activation of the BCL2 family and the three cancer cell lines, HepG2 viability was most caspase-3. Imbalanced anti-apoptotic and pro-apoptotic significantly reduced (Figure 1). Based on the results shown protein expression after a stimulus is one of the major in Figure 1, IC50 values were calculated, and further mechanisms underlying the ultimate fate of cells. We experiments were conducted at the IC50 concentration for examined BCL2, BAX, and caspase-3 protein levels in the each cell line. Neoxanthin and fucoxanthin inhibit growth of

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Figure 4. Generation of reactive oxygen species (ROS) in human cancer cells. HepG2 (A), HT-29 (B), and PC-3 (C) cells were treated with 60, 60, and 80 μM deinoxanthin for 24 h. D: The ratio of ROS generation relative to the untreated control. Values are the mean and standard deviation of three independent experiments.

Figure 5. Effect of deinoxanthin on BAX, BCL2, and caspase-3 protein expression in human cancer cells. HepG2 (A), HT-29 (B), and PC-3 (C) cells were treated with 60, 60, and 80 μM deinoxanthin for the specified time and then harvested.

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PC-3 cells by 30% within 24 h at 20 μM (12). Peridinin, a One of the apoptosis-inducing pathways is mediated carotenoid from dinoflagellates, reduces viability of human through the release of cytochrome c from mitochondria to colon cancer cells after 72 h by 60% at 20 μM (13). the cytosol, followed by activation of caspases-9 and -3. The Genistein, a soy isoflavone, reduces viability of human anti-apoptotic proteins BCL2 and BCL-XL, and the pro- prostate cancer with an IC50 value >200 μM (16). Puerarin, apoptotic protein BAX in mitochondira play important roles a flavonoid from Pueraria radix, does not inhibit viability of in cytochrome c release. β-Carotene down-regulates BCL2 colon cancer cells for the first 24 h, but inhibits by 50% at 75 protein expression, but does not affect BAX protein level μM at 72 h (17). Thus, deinoxanthin was as effective an during the induction of apoptosis of human colon cancer agent against some cancer cells as other carotenoids and cells (23). Baicalein (24) and indol-3-carbinol from Brassica phytochemicals studied previously. vegetables (25) induce apoptosis by the down-regulating The cell lines were subjected to a FACS analysis and BCL2 and BCL-XL protein levels and up-regulating BAX morphological observations to obtain additional information by inhibiting activation of protein kinase B. Yu and Li on the growth inhibition of the cancer cells by deinoxanthin. reported that puerarin induces apoptosis of HT-29 cells by The total apoptotic cell fraction increased following reducing the BCL2/BAX protein ratio and activating deinoxanthin treatment, and the apoptotic fraction was the caspase-3 (17). However, treatment of PC-3 cells with highest in HepG2 cells (Figure 2). DNA fragmentation was neoxanthin and fucoxanthin reduces BAX and BCL2 protein observed by fluorescence microscopy using Hoechst 33258 levels (12). In the present study, the levels of pro-caspase-3 staining to clearly demonstrate the apoptosis induced by and BCL2 decreased, and the BAX level increased deinoxanthin in the cancer cell lines. The cells shrank and following deinoxanthin treatment, suggesting that initiation de-formed during apoptosis. After detaching from their of intracellular events by deinoxanthin is related to neighbors, cells undergo chromosomal condensation and connected with activation of caspase-3, as the active form is internucleosomal cleavage of DNA before fragmenting into responsible for the cleavage and breakdown of several compact membrane-enclosed structures termed apoptotic cellular components related to DNA repair and regulation. bodies. Chromatin condensation occurred after a 12-h The BCL2 family of proteins also plays an important role deinoxanthin treatment and increased in a time-dependent in the regulation of apoptosis in many cellular systems, by manner (Figure 3). These results show that cell death by either inhibiting (BCL2, BCL-XL, BCL-W, BFL-1, and deinoxanthin was caused by apoptosis. MCL-1) or promoting apoptosis (BAX, BAK, BAD, BCL- The mechanism of inducing apoptosis is dependent on the Xs, BID, and HRK). Treating cancer cells with deinoxanthin carotenoid structure. In the case of β-carotene, the induction significantly altered the expression of the BCL2 family of apoptosis is coincident with an increase in intracellular members BAX and BCL2 of the mitochondrial death ROS production in HL-60 leukemia cells (18) and human pathway. These results indicate that caspase-3-dependent adenocarcinoma cells (10). Oxidative stress plays a key role apoptosis is associated with modulation of BCL2/BAX as a mediator of apoptosis, and several studies have protein ratio. supported a role for oxidative mechanisms during the Among dietary phytochemicals, carotenoids from plants induction of apoptosis. In previous studies, β-carotene and are promising cancer-preventing dietary components. In the lycopene oxidation products were found to be involved in the present study, deinoxanthin synthesized by D. radiodurans induction of apoptosis (19, 20). However, Muller et al. was investigated for its pro-apoptotic activity. We reported that a carotenoid antioxidant inhibited the demonstrated that deinoxanthin induced apoptosis in three degradation of β-carotene and significantly increased β- cancer cell lines via an enhanced level of intracellular ROS carotene-induced apoptosis in Jurkat cells (21). This result and that the apoptosis-inducing pathways were mediated indicates that the pro-oxidant effect of β-carotene was through activation of caspase-3 associated with down- unlikely to cause its pro-apoptotic activity. The effectiveness regulation of BCL2 and up-regulation of BAX proteins. of carotenoids as antioxidants and pro-oxidants is dependent The detailed mechanisms of the induction of apoptosis and on several factors including their concentration, interaction the active oxidized products of deinoxanthin will be further with other co-antioxidants, and the partial pressure of oxygen studied. (22). Kotake-Nara et al. investigated the induction of apoptosis in the PC-3 cell line by neoxanthin and Acknowledgements fucoxanthin and found that exposure to these carotenoids did not induce ROS production (12). In the present study, This study was financially supported by the Nuclear R&D program deinoxanthin treatment caused ROS to be generated in the of the Ministry of Science, ICT & Future Planning (MSIP) and by tested cell lines. In particular, ROS generation was highest Golden Seed Project, Ministry of Agriculture, Food and Rural Affairs in HepG2 cells, suggesting that deinoxanthin plays a role as (MAFRA), Ministry of Oceans and Fisheries(MOF), Rural a pro-oxidant and induces ROS and apoptosis. Development Administration (RDA) and Korea Forest Service (KFS).

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