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Dryofragin, a phloroglucinol derivative, induces apoptosis in human breast cancer MCF-7 cells through ROS-mediated mitochondrial pathway
Chemico-Biological Interactions 199 (2012) 129–136
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Dryofragin, a phloroglucinol derivative, induces apoptosis in human breast cancer MCF-7 cells through ROS-mediated mitochondrial pathway Ying Zhang a,b,1, Meng Luo a,b,1, Yuangang Zu a,b, Yujie Fu a,b,⇑, Chengbo Gu a,b, Wei Wang a,b, Liping Yao a,b, Thomas Efferth c a b c
Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany
a r t i c l e
i n f o
Article history: Received 4 March 2012 Received in revised form 8 June 2012 Accepted 9 June 2012 Available online 13 July 2012 Keywords: Dryofragin Apoptosis MCF-7 cells Reactive oxygen species
a b s t r a c t Dryofragin is a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott. In this study, the anticancer activity of dryofragin on human breast cancer MCF-7 cells was investigated. Dryofragin inhibited the growth of MCF-7 cells in a time and concentration-dependent manner. The cell viability was measured using MTT assay. After treatment with dryofragin for 72, 48 and 24 h, the IC50 values were 27.26, 37.51 and 76.10 lM, respectively. Further analyses of DNA fragmentation and Annexin V-PI double-labeling indicated an induction of apoptosis. Dryofragin-treatment MCF-7 cells had a significantly accumulation of reactive oxygen species (ROS), as well as an increased percentage of cells with mitochondrial membrane potential (MMP) disruption. These phenomena were blocked by pretreatment for 2 h of MCF-7 cells with the antioxidant compound N-acetyl-L-cysteine (NAC, 5 mM). These results speak for the involvement of a ROS-mediated mitochondria-dependent pathway in dryofragin-induced apoptosis. Western blot results showed that dryofragin inhibited Bcl-2 and induced Bax expression which led to an activation of caspases-9 and -3 in the cytosol, and further cleavage of poly ADP-ribose polymerase (PARP) in the nucleus, then induced cell apoptosis. In conclusion, the present study provides evidence that dryofragin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Ó 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The most commonly diagnosed cancer among women is breast cancer, which accounts for 28% of all new cancer cases among women [1]. Chemotherapy has been used to treat all stages of breast cancer [2]. However, chemotherapeutic agents, such as paclitaxel and anthracyclines, cause severe side effects and drug resistance in patients [3,4]. Therefore, there is continuing need to develop safe and effective anticancer drugs. Plant-derived drugs play an increasing role in cancer therapy due to their low toxicity and high efficacy. Many natural bioactive substances found in fruits, vegetables, medicinal herbs and other plants are considered as potential anti-cancer agents [5]. Clinically established natural products or their derivatives, e.g. taxanes, Vinca alkaloids, and hydroxycamptothecin, exert anti-cancer activity by
⇑ Corresponding author at: Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China. Tel./fax: +86 45182190535. E-mail address: [email protected] (Y. Fu). 1 These authors contributed equally to this work. 0009-2797/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2012.06.007
triggering apoptotic cell death and exhibit excellent anti-proliferative properties [6,7]. Apoptosis is a highly regulated process of programmed cell death that plays an important role in the maintenance of cellular homeostasis [8]. Disruption of this process represents a major contributing factor in the pathology of cancer. Apoptosis is mediated by caspases which can be activated through two pathways, the extrinsic pathway characterized by activation of cell-surface death receptors (tumor necrosis factor receptor, Fas) and the intrinsic pathway depending on the release of mitochondrial factors [9]. Caspase activation further leads to protein cleavage resulting in DNA fragmentation, chromatin condensation, and cell shrinkage. Reactive oxygen species (ROS) play a key role in mitochondriamediated apoptosis. Mitochondria are the prime source of ROS, which are byproducts of aerobic respiration [10–12]. High levels of ROS in mitochondria can result in free radical attack of membrane phospholipids and cause mitochondrial membrane depolarization. This is an irreversible step, which is associated with the release of mitochondrial factors, triggering caspase cascades [13,14]. Therefore, ROS plays an important role in mitochondriamediated apoptotic pathway.
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Dryopteris species of Aspidiaceae are rich in phloroglucinol derivatives [15]. The effects of phloroglucinol derivatives on cell apoptosis have been extensively studied, e.g. by measuring DNA cleavage, caspase activity and NF-jB-dependent gene activation [16–20]. Dryofragin is a phloroglucinol derivative from Dryopteris fragrans (L.) Schott which has been isolated by our own group [21]. Dryofragin is a ichthyotoxic compound and has anticancer activity in in vivo models such as S180 sarcoma, Lewis lung cancer and HepA liver cancer [22]. The chemical structure of dryofragin is shown in Fig. 1. The aim of the present investigation was to explore the cytotoxic activity of dryofragin towards human MCF-7 breast cancer cells and the underlying mechanisms. Our results demonstrated that dryofragin induced apoptosis by activating a ROS-mediated mitochondrial pathway, which involves with accumulation of intracellular ROS and the disruption of mitochondrial membrane potential (MMP). Altered expression of Bcl-2 and Bax proteins activated caspases-3 and -9 and the cleavage of PARP, ultimately leading to apoptosis. To the best of our knowledge, this is the first study investigating the anticancer activity and the underlying mechanisms of dryofragin in MCF-7 cells.
Cytotoxicity was expressed as the concentration of dryofragin inhibiting cell growth by 50% (IC50 value). 2.3. Quantification of apoptotic cells by flow cytometry Cells undergoing apoptosis were identified by binding of Annexin V protein to exposed phosphatidylserine (PS) residues at cell surface [24]. The extent of apoptosis was measured using Annexin V-FITC apoptosis detection kit (Beyotime Institute of Biotechnology, China) as described by the manufacturer’s instruction and analysis with flow cytometry (Partec Flow cytometry, PAS, Germany). 2.4. Morphological observation of nuclear change After treated with 30 lM dryofragin for 48 h, MCF-7 cells were washed and stained with acridine orange (AO) (200 lL, 20 lg/mL). After 10 min incubation in the dark, cells were washed with cold PBS twice, and visualized using a laser scanning confocal microscope (Nikon Eclipse TE2000-E, Tokyo, Japan) [25,26]. 2.5. Agarose gel electrophoresis for observing DNA fragmentation
2. Materials and methods 2.1. Materials and cell culture Dryofragin (purity P 98%) was isolated from Dryopteris fragrans (L.) Schott. Its chemical structure was identified in our lab [21]. A 10 mM stock solution of dryofragin was prepared in dimethyl sulfoxide (DMSO) and stored at 80 °C. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT), rhodamine123 (Rh123), NAC and propidium iodide (PI) were obtained from Sigma–Aldrich Inc. (St. Louis, MO). Deionized water was used in all experiments. The human breast cancer MCF-7 cell line was purchased from Harbin Medical University, China. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 lg/ml streptomycin. The cells were kept at 37 °C in a humidified atmosphere of 5% CO2. 2.2. Cell proliferation assays Inhibition of cell proliferation with dryofragin was measured by the MTT assay [23]. MCF-7 cells were plated in 96 wells culture plates (1 105 cells/well) separately. After 24 h incubation, cells were treated with different concentrations of dryofragin for 24, 48 or 72 h, MTT solution (5 mg/ml) was then added to each well. After incubation for 4 h, the medium was removed, and then the formazan precipitate was dissolved in 100 lL DMSO. The absorbance was measured in an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570 nm. The cell viability ratio was calculated by the following formula:
Cell viability ratio ð%Þ ¼ ðODtreated =ODcontrol Þ 100%
ð1Þ
DNA fragmentation was measured by agarose gel electrophoresis [27]. MCF-7 cells (1 106 cells/well) were seeded in 6-well plates, treated with dryofragin (0, 10, 20 and 30 lM) for 48 h. Cells were harvested and genomic DNA was extracted using a DNA isolation kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions. The DNA samples were analyzed by electrophoresis on a 1% agarose gel containing ethidium bromide and photographed by ultraviolet illumination (Image Master VDS-CL, Tokyo, Japan). 2.6. Measurement of intracellular reactive oxygen species ROS were measured by flow cytometry using an oxidant sensitive fluorescent probe dichlorofluorescein-diacetate (DCFH-DA) [29]. After 24 h incubation, cells were treated with serial dilutions of dryofragin for 12, 24, 36 and 48 h. Then, cells were harvested, washed twice with PBS, and stained with 5 lM DCFH-DA for 30 min at 37 °C in the dark. The ROS production was expressed as the mean DCFH-DA fluorescence intensity. 2.7. Analysis of mitochondrial membrane potential (DWm) MMP changes were measured by the uptake of the cationic fluorescent dye rhodamine 123 [28]. MCF-7 cells were seeded at 1 106 cells/well into 6-well plates. After 24 h incubation, cells were treated with serial dilutions of dryofragin for 12, 24, 36 and 48 h. After harvested and washed twice with PBS, the cells were then resuspended in 2 mL of fresh incubation medium containing 1.0 lM rhodamine 123 and incubated in 37 °C a thermostatic bath for 30 min with gentle shaking. Then, cells were centrifuged, washed twice with PBS, stained with 2 lg/mL propidium iodide and analyzed by flow cytometry. 2.8. Inhibitor treatment
Fig. 1. Chemical structure of dryofragin.
To confirm the effect of intracellular ROS in dryofragin-induced apoptosis, N-acetyl-L-cysteine (NAC), a common quencher of ROS, was used to inhibit the intracellular alteration of redox state [30]. MCF-7 cells were pre-incubated with 5 mM NAC for 2 h, and followed treatment with dryofragin. After 24 h, the changes of MMP and ROS production were determined by flow cytometry according to the above described methods, and the percentage of apoptotic cells was analyzed using Annexin V-FITC.
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2.9. Protein extraction and Western blotting To further evaluate the expression levels of various intracellular proteins related to apoptosis, total protein fractions were isolated and analyzed using western blotting. MCF-7 cells were treated with dryofragin for 48 h. For isolation of total protein fractions, cells were collected, washed twice with ice-cold PBS, and lysed using cell lysis buffer [20 mM Tris pH7.5, 150 mM NaCl, 1% TritonX-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1% Na3CO4, 0.5 lg/mL leupeptin, 1 mM phenylmethane-sulfonyl fluoride (PMSF)]. The lysates were collected by scraping from the plates and then centrifuged at 10,000 rpm at 4 °C for 5 min [3]. Total protein samples (20 lg) were loaded on a 12% of SDS–polyacrylamide gel for electrophoresis, and transferred onto PVDF transfer membranes (Millipore, Billerica, USA) at 0.8 mA/cm2 for 2 h. Membranes were blocked at room temperature for 2 h with blocking solution (1% BSA in PBS plus 0.05% Tween-20). Membranes were incubated overnight at 4 °C with primary antibodies (anti-b-actin, anti-Bax, anti-caspase-3, anti-caspase-9 were mouse polyclonal antibodies; anti-Bcl-2 and anti-PARP were rabbit polyclonal antibodies) at a 1:1000 dilution (Biosynthesis Biotechnology Company, Beijing, China) in blocking solution. After thrice washings in TBST for each 5 min, membranes were incubated for 1 h at room temperature with an alkaline phosphatase peroxidase-conjugated anti-mouse secondary antibody at a dilution of 1:500 in blocking solution. Detection was performed by the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Bands were recorded by a digital camera (Canon, EOS350D, Tokyo, Japan).
Fig. 2. Growth inhibition of MCF-7 cells by dryofragin as measured by MTT assay. The values for each dryofragin concentration tested represent the average (mean ± S.D.) from eight replicate wells and are representative of three separate experiments.
2.10. Statistical analysis The data were expressed as mean ± S.D. Differences between groups are assessed by one-way or two-way ANOVA, as appropriate. An analysis of ANOVA variance with a Tukey post hoc test was used for multiple comparisons. Correlations were calculated using function ReglinP and inverted Student’s t-test. All statistical calculations were performed using the STATISTICA program (StatSoft, Tulsa, OK, USA). p < 0.05 was considered significant. 3. Results 3.1. Anti-proliferation effect of dryofragin To evaluate the anti-proliferative activity of dryofragin, the viability of human cancer MCF-7 cells treated with dryofragin were determined using MTT assay. Fig. 2 shows that the growth of MCF7 cells was inhibited by dryofragin in concentration-dependent (10–100 lM) and time-dependent manner (24, 48, and 72 h). The IC50 values were 27.26 lM after 72 h treatment, 37.51 lM after 48 h, and 76.10 lM after 24 h. 3.2. Effect of dryofragin on intracellular ROS Mitochondria are the major source of ROS production, and excessive ROS accumulation may lead to oxidative stress and ultimately apoptosis [31,32]. NAC is a source of sulfhydryl groups in cells and scavenger of free radicals. It interacts with ROS such as OH and H2O2 [33] that can prevent apoptosis and promote cell survival. In order to investigate a role of ROS in dryofragin-induced apoptosis, intracellular ROS levels were examined using DCFH-DA and flow cytometry. As shown in Fig. 3A and B, treatment of MCF-7 cells with dryofragin (10–30 lM) for 12, 24, 36 and 48 h resulted in a significant elevation of intracellular ROS (from 3.56%, 5.43% 9.85% and 13.42% to 9.37%, 26.03%, 40.48% and 53.77%, p < 0.05, respec-
Fig. 3. Generation of intracellular reactive oxygen species in MCF-7 cells by dryofragin. (A) Time- and contribution-dependent change of intracellular ROS stained with DCFH-DA by the flow cytometry. (B) After pretreatment with 5 mM NAC for 2 h, the cell was incubated with 30 lM of dryofragin, and then analyzed by flow cytometry. Column represent mean values (±S.D.) of three experiments ⁄ p < 0.05 compared to control (0 lM).
tively), compared with the vehicle control. In cells treated with 5 mM NAC 2 h prior to exposure with 30 lM dryofragin for 12, 24, 36 and 48 h, the accumulation of ROS was significantly decreased from 13.42%, 26.28%, 32.34%, 53.77% to 7.76%, 14.29%, 16.75%, 34.40% (p < 0.05), respectively. 3.3. Disruption of mitochondrial membrane potential To analyze whether mitochondria were involved in dryofragininduced apoptosis, we measured MMP changes using Rh123. As
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shown in Fig. 4A and B, dryofragin led to a concentration-dependent depolarization in mitochondria. The percentage of MMP disrupted cells increased from 7.11%, 11.34%, 11.59% and 12.05% to 13.87%, 31.45%, 43.43% and 63.91% (p < 0.05), respectively. Again, pretreatment with NAC (5 mM, 2 h) prior to exposure with dryofragin (30 lM, 12, 24, 36 and 48 h) led to a decrease in the percentage of MMP disrupted cells from 12.05%, 32.47%, 37.58% and 63.91% to 9.87%, 16.47%, 18.25% and 31.14% (p < 0.05), respectively.
centages of apoptotic cells after incubation with 30 lM dryofragin (Fig. 5C and D), the percentages of early and late apoptotic cells decreased from 36.42 ± 1.34% to 26.30 ± 1.47% and 20.86 ± 1.42% to 15.12 ± 0.76% (p < 0.05), respectively. Hence, dryofragin induced apoptosis in MCF-7 cells by a ROSdependent mechanism.
3.4. Dryofragin induced apoptosis
Caspases are important regulators of apoptosis [35]. Apoptosis is executed by their coordinated actions and they are tightly regulated by pro- and anti-apoptotic Bcl-2 protein family members. We measured the protein expression of Bcl-2 and Bax in cells treated with different concentrations of dryofragin for 48 h. Fig. 6A shows a significant decrease in the levels of Bcl-2, in contrast a notable increase Bax levels. Then, we examined the activation of caspases family. As shown in Fig. 6B, treatment of MCF-7 cells with dryofragin caused a remarkable cleavage of caspases-3 and -9, indicating their activation. It is well known that the activation of caspase-3 during apoptosis can cause the cleavage of PARP, a major indicator enzyme of apoptosis. Indeed, dryofragin increased PARP cleavage in a concentration-dependent manner.
Next, we investigated whether dryofragin induces apoptotic cell death. The morphological characteristics of apoptotic cell were investigated by staining with acridine orange. Cells treated with dryofragin for 48 h showed increased percentages of cell shrinkage, condensed and fragmented chromatin, and apoptotic bodies (Fig. 5A, indicated by white arrows; 33.58 ± 1.19%, (p < 0.05). DNA fragmentation is a major biochemical event of apoptosis due to internucleosomal cleavage of DNA strands [34]. As shown in Fig. 5B, increasing concentration of dryofragin increased DNA cleavage appearing as typical ladder pattern in agarose gel electrophoresis. Apoptosis-induced DNA fragmentation was visible after treatment with 20 and 30 lM dryofragin (Fig. 5B, lanes 4 and 5). However, ladders hardly appeared in either untreated control or cells treated with 10 lM dryofragin (Fig. 5B, lanes 2 and 3). In addition, Annexin V-propidium iodide double-labeling was used to detect phosphatidylserine externalization, a hallmark of the early phase of apoptosis. After treatment with dryofragin for 48 h, the percentage of both early and late apoptotic cells increased along with the concentration of dryofragin (Fig. 5C and D). Whereas MCF-7 cells with 5 mM NAC for 2 h showed reduced per-
Fig. 4. Mitochondrial membrane potential of MCF-7 cells after treatment with dryofragin. (A) Time- and contribution-dependent change of MMP disruption stained with Rh123 by the flow cytometry. (B) After pretreatment with 5 mM NAC for 2 h, the cell was incubated with 30 lM of dryofragin, and then analyzed by flow cytometry. Column represent mean values ± S.D. of three experiments ⁄p < 0.05; compared to control (0 lM).
3.5. Effect of dryofragin on the expression of apoptosis-related proteins
4. Discussion Plant extracts bear considerable potential as anticancer agents due to their ability to inhibit tumor growth, angiogenesis, and metastasis with few side effects [31]. Several phloroglucinol derivatives from plants with pro-apoptotic capability have been identified. Examples are the cytotoxic activity of rottlerin towards human fibrosarcoma cells or of hyperforin towards leukemia cells [8,33]. Dioxinodehydroeckol isolated from Ecklonia cava was cytotoxic against human breast cancer cells by inducing apoptosis through a NF-jB dependent pathway [34]. Dryopteris species of Aspidiaceae are known to be rich in phloroglucinol derivatives. Dryofragin is one of the phloroglucinol derivatives isolated from D. fragrans (L) Schott, however, its anti-cancer effect and potential mechanisms are still not clear. Dryofragin contains two phenolic hydroxyls, which can exert different actions in modulating apoptosis. Phenolic compounds are known to act as pro-apoptotic agent, e.g. phenolic compounds derived from ginger are pro-apoptotic by a caspase-3 dependent mechanism [35]. Similar compounds activate the mitochondrial pathway by down-regulating the anti-apoptotic Bcl-2 protein and enhancing the expression of the pro-apoptotic Bax [36–38]. Another polyphenolic compound, curcumin, induced apoptosis by suppressing Bcl-2 expression and activating caspases-7 and -9 in mantle lymphoma cells [39]. The induction of apoptosis by resveratrol, a phenolic compound belonging to the class of stilbenes, was associated with increased caspase activities, decreased Bcl-2 and Bcl-XL levels, and increased Bax levels [36,40]. As phenolic hydroxyls exhibited highly pro-apoptotic activity though the mitochondria-mediated pathway, we hypothesized that dryofragin-induced apoptosis involves ROS generation and mitochondria-mediated signaling. Our results showed that the phloroglucinol derivative, dryofragin, effectively inhibited the proliferation of MCF-7 cells in a concentrationand time-dependent manner (Fig. 2). MTT is a method used to detect cell proliferation. During the process of apoptosis, the succinate dehydrogenase maintains certain activity, formazan crystallization would be still generated. After incubation 30 lM dryofragin for 48 h, the living cells account for 75%, which contained normal cell and apoptosis cell with the activity of succinate dehydrogenase. The loss of mitochondrial membrane potential (MMP) is a hallmark for apoptosis (Fig. 4A and B). Cells treated with dryofragin, the disruption of MMP was 65%, which means the early apoptotic cell
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Fig. 5. Induction of apoptosis in MCF-7 cells after dryofagin treatment for 48 h. (A) Morphology of MCF-7 cells treated with dryofragin for 48 h was observed by inverted fluorescence microscopy. (a) Untreated cells; (b) 30 lM dryofragin. (B) DNA fragmentation was analyzed by agarose gel electrophoresis. Lane 1, marker; lane 2, 0 lM dryofragin; lane 3, 10 lM dryofragin; lane 4, 20 lM dryofragin and lane 5, 30 lM dryofragin. The experiment was repeated three times and representative photographs are shown. (C) The percentage of apoptotic cells was analyzed by flow cytometry of Annexin V-propidium iodide double staining (n = 4). (a) 0 lM dryofragin; (b) 10 lM dryofragin; (c) 20 lM dryofragin; (d) 30 lM dryofragin; (e) treatment with 30 lM dryofragin and 5 mM NAC. (D) Columns represent mean values (±S.D.) of three experiments ⁄ p < 0.05 compared to control (0 lM). The experiments were repeated three times and representative photographs are shown.
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Fig. 6. Expression of apoptosis-related proteins in MCF-7 cells after dryofagin treatment for 48 h as assessed by Western blot analysis. (A) Dryofragin-mediated up-regulation of Bax and down regulation of Bcl-2. MCF-7 cells were treated with dryofragin (0, 10, 20 and 30 lM) for 48 h, respectively. (B) Effects of dryofragin on expression of caspases and PARP. MCF-7cells were treated with dryofragin (0, 10, 20 and 30 lM) for 48 h, respectively. The tests were repeated three times and representative blots are shown.
account for 65% in total living cells. We also examined various apoptotic markers in MCF-7 cells exposed to different concentration of dryofragin. After treatment with of dryofragin, the cells showed condensed and fragmented chromatin and apoptotic bodies (Fig. 5A), DNA fragmentation (Fig. 5B) and increased percentages of apoptotic cells (Fig. 5C and D). All of which suggests that apoptosis was induced by this compound. ROS were reported as a major factor causing DNA damage and apoptosis [41]. ROS produced in the mitochondria could inhibit the mitochondrial respiration chain, leading to mitochondrial membrane rupture and induction of apoptosis [42–44]. MMP disruption is a key cellular event in the mitochondrial apoptosis pathway. A reduced MMP increases the mitochondrial membrane permeability and facilitates the release of apoptotic factors. We
observed high levels of ROS, after dryofragin treatment, as well as MMP disruption in these MCF-7 cells. NAC is an antioxidant that inhibits apoptosis by scavenging ROS. The inhibition of MMP disruption (Fig. 4A and B) and the apoptotic process (Fig. 5C and D) were observed after exposing those cells in dryofragin with NAC, besides the reduced accumulation of intracellular ROS (Fig. 3A and B). After incubated with 30 lM of dryofragin for 24 or 36 h, the DCF and Rh123 fluorescence intensity dramatically changed, that means dryofragin increased the intracellular reactive oxygen species in MCF-7 cell. These results inferred that the dryofragin-induced apoptosis of MCF-7 cell via mitochondrial pathway was correlated with the generation of intracellular ROS. Hence, we tentatively conclude that dryofragin caused apoptosis by inducing ROS production
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Fig. 7. Proposed signaling pathway of dryofragin-induced apoptosis in MCF-7 cells.
and activating the mitochondrial apoptosis pathway, and other apoptotic mechanisms of dryofagin would be investigated in further study. Specific changes in the expression of apoptosis-related proteins support this conclusion, i.e. the decrease of Bcl-2 and increase of Bax expression, activation of caspases-9 and -3, as well as cleavage of PARP. Bcl-2 is an anti-apoptotic protein in mitochondria, which prevents apoptosis by suppressing oxyradical-mediated membrane damage and stabilizing MMP [45,46]. The pro-apoptotic Bax protein mediates the mitochondrial pathway by triggering the release of apoptotic factors from mitochondria after appropriate death signals [47]. The imbalance of anti-apoptotic proteins and pro-apoptotic proteins usually leads to the onset of the apoptotic process [48]. Caspases-9 and -3 play critical role in the execution of apoptosis and concomitant PARP cleavage occurs in a caspase-dependent manner. Therefore, our results suggest that dryofragin induced ROS production and activated the mitochondrial apoptosis pathway. Taken together, dryofragin induced a mitochondria-dependent apoptotic pathway via the modulation of Bax and Bcl-2 expression, resulting in MMP disruption. Loss of MMP was followed by cytochrome c release from mitochondria, leading to activation of caspases-9 and -3, and concomitant poly ADP-ribosyl polymerase (PARP) cleavage indicators of caspase-dependent apoptosis (Fig. 7). 5. Conclusion The phloroglucinol derivative dryofragin exhibited potential anti-cancer activity in human breast cancer cells through induction of apoptosis. The induction of apoptosis of dryofragin involved mitochondrial dysfunction and was tightly associated with ROS production. Our results might provide the basis for future clinical application of dryofragin treatment in breast cancer. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements The authors gratefully acknowledge the financial supports by Special Fund of Forestry Industrial Research for Public Welfare of
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Dryofragin, a phloroglucinol derivative, induces apoptosis in human breast cancer MCF-7 cells through ROS-mediated mitochondrial pathway Ying Zhang a,b,1, Meng Luo a,b,1, Yuangang Zu a,b, Yujie Fu a,b,⇑, Chengbo Gu a,b, Wei Wang a,b, Liping Yao a,b, Thomas Efferth c a b c
Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany
a r t i c l e
i n f o
Article history: Received 4 March 2012 Received in revised form 8 June 2012 Accepted 9 June 2012 Available online 13 July 2012 Keywords: Dryofragin Apoptosis MCF-7 cells Reactive oxygen species
a b s t r a c t Dryofragin is a phloroglucinol derivative extracted from Dryopteris fragrans (L.) Schott. In this study, the anticancer activity of dryofragin on human breast cancer MCF-7 cells was investigated. Dryofragin inhibited the growth of MCF-7 cells in a time and concentration-dependent manner. The cell viability was measured using MTT assay. After treatment with dryofragin for 72, 48 and 24 h, the IC50 values were 27.26, 37.51 and 76.10 lM, respectively. Further analyses of DNA fragmentation and Annexin V-PI double-labeling indicated an induction of apoptosis. Dryofragin-treatment MCF-7 cells had a significantly accumulation of reactive oxygen species (ROS), as well as an increased percentage of cells with mitochondrial membrane potential (MMP) disruption. These phenomena were blocked by pretreatment for 2 h of MCF-7 cells with the antioxidant compound N-acetyl-L-cysteine (NAC, 5 mM). These results speak for the involvement of a ROS-mediated mitochondria-dependent pathway in dryofragin-induced apoptosis. Western blot results showed that dryofragin inhibited Bcl-2 and induced Bax expression which led to an activation of caspases-9 and -3 in the cytosol, and further cleavage of poly ADP-ribose polymerase (PARP) in the nucleus, then induced cell apoptosis. In conclusion, the present study provides evidence that dryofragin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Ó 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The most commonly diagnosed cancer among women is breast cancer, which accounts for 28% of all new cancer cases among women [1]. Chemotherapy has been used to treat all stages of breast cancer [2]. However, chemotherapeutic agents, such as paclitaxel and anthracyclines, cause severe side effects and drug resistance in patients [3,4]. Therefore, there is continuing need to develop safe and effective anticancer drugs. Plant-derived drugs play an increasing role in cancer therapy due to their low toxicity and high efficacy. Many natural bioactive substances found in fruits, vegetables, medicinal herbs and other plants are considered as potential anti-cancer agents [5]. Clinically established natural products or their derivatives, e.g. taxanes, Vinca alkaloids, and hydroxycamptothecin, exert anti-cancer activity by
⇑ Corresponding author at: Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China. Tel./fax: +86 45182190535. E-mail address: [email protected] (Y. Fu). 1 These authors contributed equally to this work. 0009-2797/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2012.06.007
triggering apoptotic cell death and exhibit excellent anti-proliferative properties [6,7]. Apoptosis is a highly regulated process of programmed cell death that plays an important role in the maintenance of cellular homeostasis [8]. Disruption of this process represents a major contributing factor in the pathology of cancer. Apoptosis is mediated by caspases which can be activated through two pathways, the extrinsic pathway characterized by activation of cell-surface death receptors (tumor necrosis factor receptor, Fas) and the intrinsic pathway depending on the release of mitochondrial factors [9]. Caspase activation further leads to protein cleavage resulting in DNA fragmentation, chromatin condensation, and cell shrinkage. Reactive oxygen species (ROS) play a key role in mitochondriamediated apoptosis. Mitochondria are the prime source of ROS, which are byproducts of aerobic respiration [10–12]. High levels of ROS in mitochondria can result in free radical attack of membrane phospholipids and cause mitochondrial membrane depolarization. This is an irreversible step, which is associated with the release of mitochondrial factors, triggering caspase cascades [13,14]. Therefore, ROS plays an important role in mitochondriamediated apoptotic pathway.
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Dryopteris species of Aspidiaceae are rich in phloroglucinol derivatives [15]. The effects of phloroglucinol derivatives on cell apoptosis have been extensively studied, e.g. by measuring DNA cleavage, caspase activity and NF-jB-dependent gene activation [16–20]. Dryofragin is a phloroglucinol derivative from Dryopteris fragrans (L.) Schott which has been isolated by our own group [21]. Dryofragin is a ichthyotoxic compound and has anticancer activity in in vivo models such as S180 sarcoma, Lewis lung cancer and HepA liver cancer [22]. The chemical structure of dryofragin is shown in Fig. 1. The aim of the present investigation was to explore the cytotoxic activity of dryofragin towards human MCF-7 breast cancer cells and the underlying mechanisms. Our results demonstrated that dryofragin induced apoptosis by activating a ROS-mediated mitochondrial pathway, which involves with accumulation of intracellular ROS and the disruption of mitochondrial membrane potential (MMP). Altered expression of Bcl-2 and Bax proteins activated caspases-3 and -9 and the cleavage of PARP, ultimately leading to apoptosis. To the best of our knowledge, this is the first study investigating the anticancer activity and the underlying mechanisms of dryofragin in MCF-7 cells.
Cytotoxicity was expressed as the concentration of dryofragin inhibiting cell growth by 50% (IC50 value). 2.3. Quantification of apoptotic cells by flow cytometry Cells undergoing apoptosis were identified by binding of Annexin V protein to exposed phosphatidylserine (PS) residues at cell surface [24]. The extent of apoptosis was measured using Annexin V-FITC apoptosis detection kit (Beyotime Institute of Biotechnology, China) as described by the manufacturer’s instruction and analysis with flow cytometry (Partec Flow cytometry, PAS, Germany). 2.4. Morphological observation of nuclear change After treated with 30 lM dryofragin for 48 h, MCF-7 cells were washed and stained with acridine orange (AO) (200 lL, 20 lg/mL). After 10 min incubation in the dark, cells were washed with cold PBS twice, and visualized using a laser scanning confocal microscope (Nikon Eclipse TE2000-E, Tokyo, Japan) [25,26]. 2.5. Agarose gel electrophoresis for observing DNA fragmentation
2. Materials and methods 2.1. Materials and cell culture Dryofragin (purity P 98%) was isolated from Dryopteris fragrans (L.) Schott. Its chemical structure was identified in our lab [21]. A 10 mM stock solution of dryofragin was prepared in dimethyl sulfoxide (DMSO) and stored at 80 °C. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT), rhodamine123 (Rh123), NAC and propidium iodide (PI) were obtained from Sigma–Aldrich Inc. (St. Louis, MO). Deionized water was used in all experiments. The human breast cancer MCF-7 cell line was purchased from Harbin Medical University, China. Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 lg/ml streptomycin. The cells were kept at 37 °C in a humidified atmosphere of 5% CO2. 2.2. Cell proliferation assays Inhibition of cell proliferation with dryofragin was measured by the MTT assay [23]. MCF-7 cells were plated in 96 wells culture plates (1 105 cells/well) separately. After 24 h incubation, cells were treated with different concentrations of dryofragin for 24, 48 or 72 h, MTT solution (5 mg/ml) was then added to each well. After incubation for 4 h, the medium was removed, and then the formazan precipitate was dissolved in 100 lL DMSO. The absorbance was measured in an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570 nm. The cell viability ratio was calculated by the following formula:
Cell viability ratio ð%Þ ¼ ðODtreated =ODcontrol Þ 100%
ð1Þ
DNA fragmentation was measured by agarose gel electrophoresis [27]. MCF-7 cells (1 106 cells/well) were seeded in 6-well plates, treated with dryofragin (0, 10, 20 and 30 lM) for 48 h. Cells were harvested and genomic DNA was extracted using a DNA isolation kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions. The DNA samples were analyzed by electrophoresis on a 1% agarose gel containing ethidium bromide and photographed by ultraviolet illumination (Image Master VDS-CL, Tokyo, Japan). 2.6. Measurement of intracellular reactive oxygen species ROS were measured by flow cytometry using an oxidant sensitive fluorescent probe dichlorofluorescein-diacetate (DCFH-DA) [29]. After 24 h incubation, cells were treated with serial dilutions of dryofragin for 12, 24, 36 and 48 h. Then, cells were harvested, washed twice with PBS, and stained with 5 lM DCFH-DA for 30 min at 37 °C in the dark. The ROS production was expressed as the mean DCFH-DA fluorescence intensity. 2.7. Analysis of mitochondrial membrane potential (DWm) MMP changes were measured by the uptake of the cationic fluorescent dye rhodamine 123 [28]. MCF-7 cells were seeded at 1 106 cells/well into 6-well plates. After 24 h incubation, cells were treated with serial dilutions of dryofragin for 12, 24, 36 and 48 h. After harvested and washed twice with PBS, the cells were then resuspended in 2 mL of fresh incubation medium containing 1.0 lM rhodamine 123 and incubated in 37 °C a thermostatic bath for 30 min with gentle shaking. Then, cells were centrifuged, washed twice with PBS, stained with 2 lg/mL propidium iodide and analyzed by flow cytometry. 2.8. Inhibitor treatment
Fig. 1. Chemical structure of dryofragin.
To confirm the effect of intracellular ROS in dryofragin-induced apoptosis, N-acetyl-L-cysteine (NAC), a common quencher of ROS, was used to inhibit the intracellular alteration of redox state [30]. MCF-7 cells were pre-incubated with 5 mM NAC for 2 h, and followed treatment with dryofragin. After 24 h, the changes of MMP and ROS production were determined by flow cytometry according to the above described methods, and the percentage of apoptotic cells was analyzed using Annexin V-FITC.
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2.9. Protein extraction and Western blotting To further evaluate the expression levels of various intracellular proteins related to apoptosis, total protein fractions were isolated and analyzed using western blotting. MCF-7 cells were treated with dryofragin for 48 h. For isolation of total protein fractions, cells were collected, washed twice with ice-cold PBS, and lysed using cell lysis buffer [20 mM Tris pH7.5, 150 mM NaCl, 1% TritonX-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1% Na3CO4, 0.5 lg/mL leupeptin, 1 mM phenylmethane-sulfonyl fluoride (PMSF)]. The lysates were collected by scraping from the plates and then centrifuged at 10,000 rpm at 4 °C for 5 min [3]. Total protein samples (20 lg) were loaded on a 12% of SDS–polyacrylamide gel for electrophoresis, and transferred onto PVDF transfer membranes (Millipore, Billerica, USA) at 0.8 mA/cm2 for 2 h. Membranes were blocked at room temperature for 2 h with blocking solution (1% BSA in PBS plus 0.05% Tween-20). Membranes were incubated overnight at 4 °C with primary antibodies (anti-b-actin, anti-Bax, anti-caspase-3, anti-caspase-9 were mouse polyclonal antibodies; anti-Bcl-2 and anti-PARP were rabbit polyclonal antibodies) at a 1:1000 dilution (Biosynthesis Biotechnology Company, Beijing, China) in blocking solution. After thrice washings in TBST for each 5 min, membranes were incubated for 1 h at room temperature with an alkaline phosphatase peroxidase-conjugated anti-mouse secondary antibody at a dilution of 1:500 in blocking solution. Detection was performed by the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Bands were recorded by a digital camera (Canon, EOS350D, Tokyo, Japan).
Fig. 2. Growth inhibition of MCF-7 cells by dryofragin as measured by MTT assay. The values for each dryofragin concentration tested represent the average (mean ± S.D.) from eight replicate wells and are representative of three separate experiments.
2.10. Statistical analysis The data were expressed as mean ± S.D. Differences between groups are assessed by one-way or two-way ANOVA, as appropriate. An analysis of ANOVA variance with a Tukey post hoc test was used for multiple comparisons. Correlations were calculated using function ReglinP and inverted Student’s t-test. All statistical calculations were performed using the STATISTICA program (StatSoft, Tulsa, OK, USA). p < 0.05 was considered significant. 3. Results 3.1. Anti-proliferation effect of dryofragin To evaluate the anti-proliferative activity of dryofragin, the viability of human cancer MCF-7 cells treated with dryofragin were determined using MTT assay. Fig. 2 shows that the growth of MCF7 cells was inhibited by dryofragin in concentration-dependent (10–100 lM) and time-dependent manner (24, 48, and 72 h). The IC50 values were 27.26 lM after 72 h treatment, 37.51 lM after 48 h, and 76.10 lM after 24 h. 3.2. Effect of dryofragin on intracellular ROS Mitochondria are the major source of ROS production, and excessive ROS accumulation may lead to oxidative stress and ultimately apoptosis [31,32]. NAC is a source of sulfhydryl groups in cells and scavenger of free radicals. It interacts with ROS such as OH and H2O2 [33] that can prevent apoptosis and promote cell survival. In order to investigate a role of ROS in dryofragin-induced apoptosis, intracellular ROS levels were examined using DCFH-DA and flow cytometry. As shown in Fig. 3A and B, treatment of MCF-7 cells with dryofragin (10–30 lM) for 12, 24, 36 and 48 h resulted in a significant elevation of intracellular ROS (from 3.56%, 5.43% 9.85% and 13.42% to 9.37%, 26.03%, 40.48% and 53.77%, p < 0.05, respec-
Fig. 3. Generation of intracellular reactive oxygen species in MCF-7 cells by dryofragin. (A) Time- and contribution-dependent change of intracellular ROS stained with DCFH-DA by the flow cytometry. (B) After pretreatment with 5 mM NAC for 2 h, the cell was incubated with 30 lM of dryofragin, and then analyzed by flow cytometry. Column represent mean values (±S.D.) of three experiments ⁄ p < 0.05 compared to control (0 lM).
tively), compared with the vehicle control. In cells treated with 5 mM NAC 2 h prior to exposure with 30 lM dryofragin for 12, 24, 36 and 48 h, the accumulation of ROS was significantly decreased from 13.42%, 26.28%, 32.34%, 53.77% to 7.76%, 14.29%, 16.75%, 34.40% (p < 0.05), respectively. 3.3. Disruption of mitochondrial membrane potential To analyze whether mitochondria were involved in dryofragininduced apoptosis, we measured MMP changes using Rh123. As
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shown in Fig. 4A and B, dryofragin led to a concentration-dependent depolarization in mitochondria. The percentage of MMP disrupted cells increased from 7.11%, 11.34%, 11.59% and 12.05% to 13.87%, 31.45%, 43.43% and 63.91% (p < 0.05), respectively. Again, pretreatment with NAC (5 mM, 2 h) prior to exposure with dryofragin (30 lM, 12, 24, 36 and 48 h) led to a decrease in the percentage of MMP disrupted cells from 12.05%, 32.47%, 37.58% and 63.91% to 9.87%, 16.47%, 18.25% and 31.14% (p < 0.05), respectively.
centages of apoptotic cells after incubation with 30 lM dryofragin (Fig. 5C and D), the percentages of early and late apoptotic cells decreased from 36.42 ± 1.34% to 26.30 ± 1.47% and 20.86 ± 1.42% to 15.12 ± 0.76% (p < 0.05), respectively. Hence, dryofragin induced apoptosis in MCF-7 cells by a ROSdependent mechanism.
3.4. Dryofragin induced apoptosis
Caspases are important regulators of apoptosis [35]. Apoptosis is executed by their coordinated actions and they are tightly regulated by pro- and anti-apoptotic Bcl-2 protein family members. We measured the protein expression of Bcl-2 and Bax in cells treated with different concentrations of dryofragin for 48 h. Fig. 6A shows a significant decrease in the levels of Bcl-2, in contrast a notable increase Bax levels. Then, we examined the activation of caspases family. As shown in Fig. 6B, treatment of MCF-7 cells with dryofragin caused a remarkable cleavage of caspases-3 and -9, indicating their activation. It is well known that the activation of caspase-3 during apoptosis can cause the cleavage of PARP, a major indicator enzyme of apoptosis. Indeed, dryofragin increased PARP cleavage in a concentration-dependent manner.
Next, we investigated whether dryofragin induces apoptotic cell death. The morphological characteristics of apoptotic cell were investigated by staining with acridine orange. Cells treated with dryofragin for 48 h showed increased percentages of cell shrinkage, condensed and fragmented chromatin, and apoptotic bodies (Fig. 5A, indicated by white arrows; 33.58 ± 1.19%, (p < 0.05). DNA fragmentation is a major biochemical event of apoptosis due to internucleosomal cleavage of DNA strands [34]. As shown in Fig. 5B, increasing concentration of dryofragin increased DNA cleavage appearing as typical ladder pattern in agarose gel electrophoresis. Apoptosis-induced DNA fragmentation was visible after treatment with 20 and 30 lM dryofragin (Fig. 5B, lanes 4 and 5). However, ladders hardly appeared in either untreated control or cells treated with 10 lM dryofragin (Fig. 5B, lanes 2 and 3). In addition, Annexin V-propidium iodide double-labeling was used to detect phosphatidylserine externalization, a hallmark of the early phase of apoptosis. After treatment with dryofragin for 48 h, the percentage of both early and late apoptotic cells increased along with the concentration of dryofragin (Fig. 5C and D). Whereas MCF-7 cells with 5 mM NAC for 2 h showed reduced per-
Fig. 4. Mitochondrial membrane potential of MCF-7 cells after treatment with dryofragin. (A) Time- and contribution-dependent change of MMP disruption stained with Rh123 by the flow cytometry. (B) After pretreatment with 5 mM NAC for 2 h, the cell was incubated with 30 lM of dryofragin, and then analyzed by flow cytometry. Column represent mean values ± S.D. of three experiments ⁄p < 0.05; compared to control (0 lM).
3.5. Effect of dryofragin on the expression of apoptosis-related proteins
4. Discussion Plant extracts bear considerable potential as anticancer agents due to their ability to inhibit tumor growth, angiogenesis, and metastasis with few side effects [31]. Several phloroglucinol derivatives from plants with pro-apoptotic capability have been identified. Examples are the cytotoxic activity of rottlerin towards human fibrosarcoma cells or of hyperforin towards leukemia cells [8,33]. Dioxinodehydroeckol isolated from Ecklonia cava was cytotoxic against human breast cancer cells by inducing apoptosis through a NF-jB dependent pathway [34]. Dryopteris species of Aspidiaceae are known to be rich in phloroglucinol derivatives. Dryofragin is one of the phloroglucinol derivatives isolated from D. fragrans (L) Schott, however, its anti-cancer effect and potential mechanisms are still not clear. Dryofragin contains two phenolic hydroxyls, which can exert different actions in modulating apoptosis. Phenolic compounds are known to act as pro-apoptotic agent, e.g. phenolic compounds derived from ginger are pro-apoptotic by a caspase-3 dependent mechanism [35]. Similar compounds activate the mitochondrial pathway by down-regulating the anti-apoptotic Bcl-2 protein and enhancing the expression of the pro-apoptotic Bax [36–38]. Another polyphenolic compound, curcumin, induced apoptosis by suppressing Bcl-2 expression and activating caspases-7 and -9 in mantle lymphoma cells [39]. The induction of apoptosis by resveratrol, a phenolic compound belonging to the class of stilbenes, was associated with increased caspase activities, decreased Bcl-2 and Bcl-XL levels, and increased Bax levels [36,40]. As phenolic hydroxyls exhibited highly pro-apoptotic activity though the mitochondria-mediated pathway, we hypothesized that dryofragin-induced apoptosis involves ROS generation and mitochondria-mediated signaling. Our results showed that the phloroglucinol derivative, dryofragin, effectively inhibited the proliferation of MCF-7 cells in a concentrationand time-dependent manner (Fig. 2). MTT is a method used to detect cell proliferation. During the process of apoptosis, the succinate dehydrogenase maintains certain activity, formazan crystallization would be still generated. After incubation 30 lM dryofragin for 48 h, the living cells account for 75%, which contained normal cell and apoptosis cell with the activity of succinate dehydrogenase. The loss of mitochondrial membrane potential (MMP) is a hallmark for apoptosis (Fig. 4A and B). Cells treated with dryofragin, the disruption of MMP was 65%, which means the early apoptotic cell
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Fig. 5. Induction of apoptosis in MCF-7 cells after dryofagin treatment for 48 h. (A) Morphology of MCF-7 cells treated with dryofragin for 48 h was observed by inverted fluorescence microscopy. (a) Untreated cells; (b) 30 lM dryofragin. (B) DNA fragmentation was analyzed by agarose gel electrophoresis. Lane 1, marker; lane 2, 0 lM dryofragin; lane 3, 10 lM dryofragin; lane 4, 20 lM dryofragin and lane 5, 30 lM dryofragin. The experiment was repeated three times and representative photographs are shown. (C) The percentage of apoptotic cells was analyzed by flow cytometry of Annexin V-propidium iodide double staining (n = 4). (a) 0 lM dryofragin; (b) 10 lM dryofragin; (c) 20 lM dryofragin; (d) 30 lM dryofragin; (e) treatment with 30 lM dryofragin and 5 mM NAC. (D) Columns represent mean values (±S.D.) of three experiments ⁄ p < 0.05 compared to control (0 lM). The experiments were repeated three times and representative photographs are shown.
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Fig. 6. Expression of apoptosis-related proteins in MCF-7 cells after dryofagin treatment for 48 h as assessed by Western blot analysis. (A) Dryofragin-mediated up-regulation of Bax and down regulation of Bcl-2. MCF-7 cells were treated with dryofragin (0, 10, 20 and 30 lM) for 48 h, respectively. (B) Effects of dryofragin on expression of caspases and PARP. MCF-7cells were treated with dryofragin (0, 10, 20 and 30 lM) for 48 h, respectively. The tests were repeated three times and representative blots are shown.
account for 65% in total living cells. We also examined various apoptotic markers in MCF-7 cells exposed to different concentration of dryofragin. After treatment with of dryofragin, the cells showed condensed and fragmented chromatin and apoptotic bodies (Fig. 5A), DNA fragmentation (Fig. 5B) and increased percentages of apoptotic cells (Fig. 5C and D). All of which suggests that apoptosis was induced by this compound. ROS were reported as a major factor causing DNA damage and apoptosis [41]. ROS produced in the mitochondria could inhibit the mitochondrial respiration chain, leading to mitochondrial membrane rupture and induction of apoptosis [42–44]. MMP disruption is a key cellular event in the mitochondrial apoptosis pathway. A reduced MMP increases the mitochondrial membrane permeability and facilitates the release of apoptotic factors. We
observed high levels of ROS, after dryofragin treatment, as well as MMP disruption in these MCF-7 cells. NAC is an antioxidant that inhibits apoptosis by scavenging ROS. The inhibition of MMP disruption (Fig. 4A and B) and the apoptotic process (Fig. 5C and D) were observed after exposing those cells in dryofragin with NAC, besides the reduced accumulation of intracellular ROS (Fig. 3A and B). After incubated with 30 lM of dryofragin for 24 or 36 h, the DCF and Rh123 fluorescence intensity dramatically changed, that means dryofragin increased the intracellular reactive oxygen species in MCF-7 cell. These results inferred that the dryofragin-induced apoptosis of MCF-7 cell via mitochondrial pathway was correlated with the generation of intracellular ROS. Hence, we tentatively conclude that dryofragin caused apoptosis by inducing ROS production
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Fig. 7. Proposed signaling pathway of dryofragin-induced apoptosis in MCF-7 cells.
and activating the mitochondrial apoptosis pathway, and other apoptotic mechanisms of dryofagin would be investigated in further study. Specific changes in the expression of apoptosis-related proteins support this conclusion, i.e. the decrease of Bcl-2 and increase of Bax expression, activation of caspases-9 and -3, as well as cleavage of PARP. Bcl-2 is an anti-apoptotic protein in mitochondria, which prevents apoptosis by suppressing oxyradical-mediated membrane damage and stabilizing MMP [45,46]. The pro-apoptotic Bax protein mediates the mitochondrial pathway by triggering the release of apoptotic factors from mitochondria after appropriate death signals [47]. The imbalance of anti-apoptotic proteins and pro-apoptotic proteins usually leads to the onset of the apoptotic process [48]. Caspases-9 and -3 play critical role in the execution of apoptosis and concomitant PARP cleavage occurs in a caspase-dependent manner. Therefore, our results suggest that dryofragin induced ROS production and activated the mitochondrial apoptosis pathway. Taken together, dryofragin induced a mitochondria-dependent apoptotic pathway via the modulation of Bax and Bcl-2 expression, resulting in MMP disruption. Loss of MMP was followed by cytochrome c release from mitochondria, leading to activation of caspases-9 and -3, and concomitant poly ADP-ribosyl polymerase (PARP) cleavage indicators of caspase-dependent apoptosis (Fig. 7). 5. Conclusion The phloroglucinol derivative dryofragin exhibited potential anti-cancer activity in human breast cancer cells through induction of apoptosis. The induction of apoptosis of dryofragin involved mitochondrial dysfunction and was tightly associated with ROS production. Our results might provide the basis for future clinical application of dryofragin treatment in breast cancer. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements The authors gratefully acknowledge the financial supports by Special Fund of Forestry Industrial Research for Public Welfare of
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