J Cancer Res Clin Oncol (2015) 141:255–268 DOI 10.1007/s00432-014-1819-8
ORIGINAL ARTICLE – CANCER RESEARCH
Dieckol, isolated from the edible brown algae Ecklonia cava, induces apoptosis of ovarian cancer cells and inhibits tumor xenograft growth Ji‑Hye Ahn · Yeong‑In Yang · Kyung‑Tae Lee · Jung‑Hye Choi
Received: 18 April 2014 / Accepted: 28 August 2014 / Published online: 13 September 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Ecklonia cava is an abundant brown alga and has been reported to possess various bioactive compounds having anti-inflammatory effect. However, the anticancer effects of dieckol, a major active compound in E. cava, are poorly understood. In the present study, we investigated the anti-tumor activity of dieckol and its molecular mechanism in ovarian cancer cells and in a xenograft mouse model. Methods MTT assay, PI staining, and PI and Annexin double staining were performed to study cell cytotoxicity, cell cycle distribution, and apoptosis. We also investigated reactive oxygen species (ROS) production and protein expression using flow cytometry and Western blot analysis, respectively. Anti-tumor effects of dieckol were evaluated in SKOV3 tumor xenograft model. Results We found that the E. cava extract and its phlorotannins have cytotoxic effects on A2780 and SKOV3 ovarian cancer cells. Dieckol induced the apoptosis of SKOV3 cells and suppressed tumor growth without any significant adverse effect in the SKOV3-bearing mouse model. Dieckol triggered the activation of caspase-8, caspase-9, and caspase-3, and pretreatment with caspase inhibitors neutralized the pro-apoptotic activity of dieckol. Electronic supplementary material The online version of this article (doi:10.1007/s00432-014-1819-8) contains supplementary material, which is available to authorized users. J.-H. Ahn · Y.-I. Yang · K.-T. Lee · J.-H. Choi Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul, Republic of Korea J.-H. Ahn · Y.-I. Yang · J.-H. Choi (*) Division of Molecular Biology, College of Pharmacy, Kyung Hee University, Dongdaemun‑Gu, Hoegi‑Dong, Seoul 130‑701, Republic of Korea e-mail: [email protected]
Furthermore, treatment with dieckol caused mitochondrial dysfunction and suppressed the levels of anti-apoptotic proteins. We further demonstrated that dieckol induced an increase in intracellular ROS, and the antioxidant N-acetyll-cysteine (NAC) significantly reversed the caspase activation, cytochrome c release, Bcl-2 downregulation, and apoptosis that were caused by dieckol. Moreover, dieckol inhibited the activity of AKT and p38, and overexpression of AKT and p38, at least in part, reversed dieckol-induced apoptosis in SKOV3 cells. Conclusion These data suggest that dieckol suppresses ovarian cancer cell growth by inducing caspase-dependent apoptosis via ROS production and the regulation of AKT and p38 signaling. Keywords Apoptosis · Ecklonia cava · Dieckol · Ovarian cancer · Reactive oxygen species · Tumor xenograft model
Introduction Ovarian epithelial cancer affects approximately 204,000 women and is responsible for approximately 125,000 deaths per year worldwide (Parkin et al. 2005). In most cases, patients present with stage III or IV cancer, which have 5-year survival rates of 28 and 16 %, respectively. The standard management for advanced ovarian cancer is cytoreductive surgery followed by taxane/platinum-based chemotherapy (Conte et al. 1999). However, patients with advanced or more aggressive tumors often experience chemoresistance and recurrence, leading to a poor longterm survival rate. In addition, side effects such as nephrotoxicity, ototoxicity (hearing loss and balance disturbance), and hepatotoxicity are common among patients treated with the conventional chemotherapeutic agents (Ozcelik
et al. 2010). Thus, there is an urgent need for new therapeutic agents that provide quality of life and higher survival rates for patients with ovarian cancer. Ecklonia cava is an edible brown alga found along the Pacific coast around Japan and Korea. Brown algae are a rich source of nutrition, including minerals, dietary fiber, polysaccharides, and peptides, and are a popular food ingredient, a supplement for animal feed, and a health food supplement in East Asia (Kim et al. 2008; Lee et al. 2010; Wijesinghe and Jeon 2012). Recently, this seaweed has attracted extensive interest due to its novel and highly bioactive components, which have multiple biological and pharmacological activities. In fact, E. cava has been identified as a potential producer of a wide spectrum of natural substances, such as carotenoids, fucoidans, and phlorotannins, which show different biological activities in vital industrial applications, including pharmaceutical, nutraceutical, cosmeceutical, and functional foods (Lee et al. 2012a; Wijesinghe and Jeon 2012). Brown algal phlorotannins, with a unique phloroglucinol (1,3,5-trihydroxybenzene) structure, represent a large and commonly studied class of marine secondary metabolites. The phlorotannins eckol (a closed-chain trimer of phloroglucinol), 6,6′-bieckol (a hexamer), dieckol (a hexamer), and phlorofucofuroeckol (a pentamer) have been isolated from the Ecklonia species and have been demonstrated to possess numerous biological activities, including antibacterial (Nagayama et al. 2002), anti-inflammatory (Shin et al. 2006; Yang et al. 2012), antioxidant (Kang et al. 2004), plasmin-activating (Fukuyama et al. 1989), anti‐matrix metalloproteinase (MMP) (Joe et al. 2006), and anti‐HIV (Artan et al. 2008) activities in both in vitro and in vivo model studies. In addition, some anticancer activities of phlorotannins have also been reported (Athukorala et al. 2006; Kong et al. 2009). For example, dieckol inhibited the migration and invasion of B16 mouse melanoma and HT1080 human fibrosarcoma cells (Park and Jeon 2012; Park et al. 2012; Zhang et al. 2011). However, the effects of dieckol on cancer cell growth are poorly understood, and the anticancer effect of dieckol has never been investigated in an in vivo model. Thus, in the present study, we investigated the effects of dieckol on apoptosis induction in human ovarian cancer cells and further examined its effect on tumor growth in a nude mouse model.
Materials and methods
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Island, South Korea, was extracted by ethanol, and the total polyphenol content of the extract as phloroglucinol (anhydrous) equivalents was 92 %. The phlorotannins in the extract were found to include dieckol (16.6 %), 6,6′-bieckol (3.6 %), 2-O-(2,4,6-trihydroxyphenyl)-6,6′-bieckol (3.3 %), 8,8′-bieckol + 7-phloroeckol (9.3 %), phlorofucofuroeckol A (5.4 %), eckol (2.8 %), 2-phloroeckol (2.5 %), phlorotannin A (2.3 %), and fucofuroeckol A (2.5 %), as determined by HPLC [Waters, column: Spherisorb S10ODS2 column (20 × 250 mm2); eluent: 30 % aqueous MeOH; flow rate: 3.5 mL/min]. RPMI 1640, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Molecular Probes Inc (Eugene, OR, USA), propidium iodide (PI) was purchased from Sigma Chemical (St. Louis, MO, USA), and phenylmethylsulfonyl fluoride (PMSF), annexin V-fluorescein isothiocyanate (FITC), and antibodies for caspase-8 were purchased from BD Biosciences (San Jose, CA, USA). Hydroethidine (HEt) was obtained from Assay BioTech (San Francisco, CA, USA), and dichlorofluorescein diacetate (DCFH-DA) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The pcDNA3-Akt-Myr and pMT3-p38 were obtained from Addgene (Cambridge, MA, USA). The anticaspase-3, Bcl-2, and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the caspase-9 antibody was obtained from Cell Signaling (Beverly, MA, USA). z-VAD-fmk and z-DEVD-fmk were purchased from Calbiochem (Bad Soden, Germany). Cell culture and MTT assay The ovarian cancer cell lines SKOV3 and A2780 were purchased from American Type Culture Collection (ATCC). The cells were cultured in RPMI 1640 supplemented with 5 % fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin sulfate (100 μg/mL). The cytotoxicity was assessed using the MTT assay. Briefly, the cells (5 × 104) were seeded in each well containing 50 μL of RPMI medium in a 96-well plate. After 24 h, various concentrations of dieckol were added. After 48 h, 25 μL of MTT (5 mg/mL stock solution) was added, and the plates were incubated for an additional 4 h. The medium was discarded, and the formazan blue, which was formed in the cells, was dissolved in 50 μL of DMSO. The optical density was measured at 540 nm using a microplate spectrophotometer (SpectraMax; Molecular Devices, Sunnyvale, CA, USA).
Materials Propidium iodide (PI) staining for cell cycle analysis The Ecklonia cava extract and dieckol (>99 % purity) used for this study were kindly supplied by Botamedi, Inc. (Jeju, South Korea). The Ecklonia cava, collected from Jeju
On the day of collection, the cells were harvested and washed twice with ice-cold PBS. The cells were fixed
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and permeabilized with 70 % ice-cold ethanol at 4 °C for 1 h. The cells were washed once with PBS and resuspended in a staining solution containing propidium iodide (50 μg/mL) and RNase A (250 μg/mL). The cell suspensions were incubated for 30 min at room temperature in the dark and then subjected to fluorescenceactivated cell sorting (FACS) cater-plus flow cytometry (Becton–Dickinson, Heidelberg, Germany) using 10,000 cells per group. Annexin V and PI double staining for apoptosis analysis During apoptosis, the exposure of phosphatidylserine on the exterior surface of the plasma membrane can be detected by the binding of fluoresceinated annexin V (annexin V-FITC). This assay is combined with the analysis of the exclusion of the plasma membrane integrity probe PI. To double stain with annexin V and PI, the cells were suspended in 100 μL of binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and stained with 5 μL of FITC-conjugated annexin V and 5 μL of PI (50 μg/mL). The mixture was incubated for 15 min at room temperature in the dark and analyzed using a FACS cater-plus flow cytometer. Caspases activity assay Activities of caspases induced by dieckol were assayed using a colorimetric assay kit (Calbiochem) according to the manufacturer’s protocol. Briefly, the cells were harvested and lysed in a lysis buffer for 30 min on an ice. The lysed cells were centrifuged at 10,000×g for 10 min, and 10 μg of the protein was incubated with 80 μg of an assay buffer and 10 μL of the substrate (caspase-3; Ac-DEVDAFC, Calbiochem, caspase-8; Ac-IETD-AFC, Calbiochem, and caspase-9; Ac-LEHD-AFC, Calbiochem, respectively) at 37 °C for 2 h. The reactions of mixture were measured spectrophotometrically at a wavelength of 405 nm using a microplate spectrophotometer (SpectraMax; Molecular Devices, Sunnyvale, CA, USA). Measurement of reactive oxygen species (ROS) The intracellular accumulation of ROS was determined using the fluorescent probe DCFH-DA and HEt. DCFHDA is commonly used to measure H2O2. The cells were collected by centrifugation 30 min before treatment with the cytotoxic agents, resuspended in PBS, and loaded with 20 μM DCFH-DA. The fluorescence was measured at the desired time intervals by flow cytometry. To measure superoxide (O2·), HEt, which reacts with superoxide radical to form 2-hydroxyethidium, has been used as previously described (Balcerczyk et al. 2005; Pelicano et al. 2003).
Briefly, after treatment, the cells were incubated with 1 μM HEt (O2·−) for 30 min, then washed with PBS, and immediately analyzed by flow cytometry. Cell fractionation and Western blot analysis The cells were washed with ice-cold PBS and extracted using a mitochondrial fractionation kit (ActiveMotif, Carlsbad, CA, USA). Cells were collected by centrifugation (600×g, 5 min, 4 °C). The cells were then washed twice with ice-cold PBS and centrifuged (600×g, 5 min, 4 °C). The cell pellet obtained was then resuspended in ice-cold cytosolic buffer for 15 min on ice. The cells were then homogenized with a glass Dounce and a B-type pestle (80 strokes). The homogenates were spun at 10,000×g for 20 min at 4 °C, and the supernatant (cytosolic fraction) was removed while taking care to avoid disrupting the pellet. The resulting pellet (mitochondrial fraction) was resuspended in complete mitochondrial buffer. To obtain total cell protein extracts, the cells were washed with icecold PBS and extracted in protein lysis buffer (Intron, Seoul, South Korea). The protein concentration was determined using the Bradford assay. The protein samples from the cell lysates were mixed with an equal volume of 5 × SDS sample buffer, boiled for 4 min, and then separated on 10–15 % SDS-PAGE gels. After electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5 % non-fat dry milk for 30 min, washed, and incubated overnight at 4 °C with specific primary antibodies in Trisbuffered saline (TBS) containing Tween-20 (0.1 %). The primary antibodies were removed by washing the membranes three times in TBS-T, and then, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (1:1,000–2,000). Following three washes in TBS-T, immunopositive bands were visualized by enhanced chemiluminescence and exposed to ImageQuant LAS-4000 (Fujifilm Life Science, Tokyo, Japan). Analysis of Δψm Changes in Δψm were monitored by flow cytometric analysis as described previously (Cao et al. 2006; Kim et al. 2012; Li et al. 2014). The cells were incubated with 50 nM DiOC6 for 30 min, washed twice with PBS, and analyzed by flow cytometric analysis (Becton–Dickinson, Heidelberg, Germany) with excitation and emission settings of 484 and 500 nm, respectively. To ensure that the DiOC6 uptake was specific for Δψm, we also treated the cells with 100 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The CCCP was used as a reference depolarizing agent.
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Table 1 Cytotoxic activity of E. cava extract and its components in A2780 and SKOV3 cells in vitro IC50a A2780 E. cava extract (μg/mL) Dieckol (μM) 6,6′-Bieckol (μM) 7-Phloroeckol (μM) 8,8-Bieckol (μM) Phlorofucofuroeckol A (μM) Cisplatin (μM)
84.3 ± 0.016
99.6 ± 0.050
77.31 ± 0.017 89.18 ± 0.098
92.7 ± 0.013 96.39 ± 0.023
98.14 ± 0.021 80.09 ± 0.016 137.77 ± 0.036
169.08 ± 0.012 98.08 ± 0.061 >200
3.98 ± 0.019
12.94 ± 0.027
IC50 is defined as the concentration that results in a 50 % decrease in the number of cells compared to that of the control cultures. The values represent the means of the results from three independent experiments with similar patterns. Cisplatin is treated as the positive control
Ovarian tumor xenografts BALB/c athymic female nude mice (n = 20) weighing 20–25 g, from NARA Biotech (Seoul, South Korea), were used for the studies. Ovarian carcinoma was induced by subcutaneously (s.c.) inoculating 5 × 106 SKOV3 cells (100 μL) into the flank of each mouse, and tumors were allowed to grow for 1 week. The tumorbearing mice were randomly divided into four groups (5 mice/group). For 4 consecutive weeks, the experimental group of mice received dieckol (50 and 100 mg/ kg) or cisplatin (3 mg/kg) three times per week (Monday, Wednesday, and Friday) throughout the study. Just before administration, the dieckol was prepared in DMSO and diluted tenfold in a formulation containing a final concentration of 0.1 % Tween-80. The tumor size was measured using calipers, and the tumor volume was estimated using the following formula: tumor volume (mm3) = 1/2 (L × W2), where L is the length and W is the width of the tumor. Analysis of the serum biochemical parameters Blood was drawn from the inferior vena cava into a heparin-coated tube, and the serum was obtained by centrifuging the blood at 15,000g for 15 min at 4 °C. Serum creatinine, blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using a colorimetric assay kit (Asan Pharmaceutical, Seoul, South Korea) according to the manufacturer’s instructions. The serum creatinine and BUN levels were measured as indicators of the kidney function, while the ALT and AST levels were measured to evaluate the liver function.
Statistical analysis The data are presented as the mean ± SD. The differences between the groups were identified using Student’s unpaired t test.
Results Ecklonia cava extract and phlorotannins inhibit ovarian cancer cell growth To examine the effect of E. cava extract on the growth of ovarian cancer cells, we evaluated the IC50 in ovarian cancer A2780 and SKOV3 cells using MTT assays. As shown in Table 1, the IC50 values of E. cava extract in A2780 and SKOV3 cells were 84.3 and 99.6 μg/mL, respectively, suggesting a possible growth inhibitory effect of phlorotannins found in the extract. In this regard, the cytotoxic effects of five major phlorotannins (phlorofucofuroeckol, dieckol, 6,6′-bieckol, 7-phloroeckol, and 8,8′-bieckol) were investigated in A2780 and SKVO3 cells. Among them, dieckol and bieckols (6,6′-bieckol and 8,8′-bieckol) showed a certain degree of cytotoxic activity in both A2780 and SKOV3 cells, with an IC50 range of 80–100 μM. Dieckol (Fig. 1a), a major component (16.6 %) of the E. cava extract used here, is known to have various biological activities (Lee et al. 2012b; Park et al. 2010, 2012; Zhang et al. 2011). Thus, we further investigated the cell growth inhibitory effect of dieckol on SKOV3 cells. Exponentially growing SKOV3 cells were exposed to various concentrations of dieckol for 1, 3, 5, and 7 days, and their growth was monitored. A significant decrease in cell growth was observed in the cells treated with dieckol (Fig. 1b). Dieckol induces apoptosis in SKOV3 cells To determine whether the growth inhibitory effect was associated with the induction of cell cycle arrest and/or apoptosis, the distribution of cells in the different phases of the cell cycle and the externalization of phosphatidylserine (PS) were analyzed using flow cytometry. As shown in Fig. 1c, the dieckol treatment increased the fractionation of the nuclei accumulated at sub-G1 in SKOV3 cells in a dosedependent manner, but it failed to induce cell cycle arrest, one mechanism of growth inhibition. To confirm dieckolinduced apoptosis, an annexin V-FITC staining assay was performed with SKOV3 cells. Treatment with dieckol significantly increased the population of annexin V-positive cells (apoptotic cells) in the right quadrants of flow cytometry graphs in a time- and dose-dependent manner (Fig. 1d). These results indicate that the dieckol-induced growth
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Fig. 1 Effect of dieckol on the induction of apoptosis in SKOV3 cells. a Chemical structure of dieckol, a dimeric compound of eckol. b SKOV3 cells were incubated with or without dieckol (60, 80, 100, and 120 μM) for the indicated times (1, 3, 5, and 7 days). MTT assays were performed to measure the cell growth. The data are presented as the mean ± SD of the results obtained from three independent experiments. *P