FCT 7930
No. of Pages 8, Model 5G
6 May 2014 Food and Chemical Toxicology xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox 5 6
Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway
3 4 7 8 9 10 11 12 1 2 4 6 15 16 17 18 19 20 21 22 23 24 25
Q1
Wei-Dong Ma a,1, Yong-Peng Zou b,1, Peng Wang a, Xiao-Hui Yao a, Yao Sun a, Ming-Hui Duan a, Yu-Jie Fu a,⇑, Bo Yu b,⇑ a b
Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China The 2nd Affiliated Hospital of Harbin Medical University, Harbin 150001, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 17 October 2013 Accepted 8 April 2014 Available online xxxx
Chimaphilin, 2,7-dimethyl-1,4-naphthoquinone, is extracted from pyrola [Passiflora incarnata Fisch.]. In this study, the anticancer activity and underlying mechanisms of chimaphilin toward human breast cancer MCF-7 cells are firstly investigated. Chimaphilin could inhibit the viability of MCF-7 cells in a concentration-dependent manner, and the IC50 value was 43.30 lM for 24 h. Chimaphilin markedly induced apoptosis through the investigation of characteristic apoptotic morphological changes, nuclear DNA fragmentation, annexin V-FITC/propidium iodide (PI) double staining. Flow cytometry assay revealed that chimaphilin triggered a significant generation of ROS and disruption of mitochondrial membrane potential. Additionally, western blotting assay showed that chimaphilin suppressed Bcl-2 level and enhanced Bad level, then activated caspase-9 and caspase-3, and further activated the poly ADP-ribose polymerase (PARP), finally induced cell apoptosis involving the mitochondrial pathway. Furthermore, free radical scavengers N-acetyl-L-cysteine (NAC) pretreatment test testified that chimaphilin could increase the generation of ROS, then induce cell apoptosis. In general, the present results demonstrated that chimaphilin induced apoptosis in human breast cancer MCF-7 cells via a ROS-mediated mitochondrial pathway. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Chimaphilin Apoptosis Reactive oxygen species Mitochondrial MCF-7
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
44 45
1. Introduction
46
Cancer is one of the major diseases leading to deaths in the world (Natarajan et al., 2011). In the developed world, breast cancer is one of the most common types of cancers among women, which accounts for 15–20% of all cancer deaths in the mid-1990s (Noori and Hassan, 2012). Chemotherapy and radiotherapy are
47 48 49 50
0
0
Abbreviations: AO, acridine orange; DCFH-DA, 2 ,7 -dichlorofluorescin diacetate; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl; MCF-7 cells, human breast cancer cell line; MMP, mitochondrial membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAC, N-acety-L-cystein; ROS, reactive oxygen species; PI, propidium iodide. ⇑ Corresponding authors. Address: State Engineering Laboratory of Bio-Resource Eco-Utilization, Northeast Forestry University, Hexing Road, Harbin 150040, PR China. Tel./fax: +86 451 82190535 (Y. Fu). Address: Key Laboratory of Myocardial Ischemia, Ministry of Education, The 2nd Affiliated Hospital of Harbin Medical University, Xuefu Road, Harbin 150001, PR China. Tel./fax: +86 451 86297220 (B. Yu). E-mail addresses: [email protected] (Y.-J. Fu), [email protected] (B. Yu).
1
main therapeutic method of cancer at present. However, chemotherapy and radiotherapy were generally expensive for patients. Additionally, chemotherapy generally leads to drug resistance and severe side effects for patients. Therefore, it is necessary to develop high efficient natural chemotherapeutic agents with low cost, low drug resistance and side effects. Apoptosis is a process of genetically programmed cell death that consists of a cascade of molecular events in stimulated cells (Vinatier et al., 1996). Apoptosis is triggered by biological and physical signals, such as chemical reagents, radiation, free radicals, and infection of virus et al. (Sun, 1999; Peter et al., 1997). The mechanism of apoptosis is involved in an energy-dependent cascade of molecular events. To date, the extrinsic death receptor pathway and the intrinsic mitochondrial pathway are two main apoptotic pathways (Elmore, 2007). In apoptotic cells, over-expression of intracellular reactive oxygen species (ROS) is a main cause of leading to DNA and protein damage. ROS, which consists of some radical species and non-radicals hydrogen peroxide, plays a key role in metabolism, oxidative stress, signal transduction, and apoptosis in cells (Apel, 2004; Simon et al., 2000). Generation of ROS can directly activate the mitochondrial permeability transition and
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.fct.2014.04.014 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 2
W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
110
result in loss of mitochondrial membrane potential (MMP) and the membrane integrity (Crompton et al., 2002). Subsequently, cytochrome C is released from mitochondria into the cytosol and activates the caspase cascades contributing to apoptosis of cells. Chimaphilin, 2,7-dimethyl-1,4-naphthoquinone, was isolated from pyrola [Passiflora incarnata Fisch.] in our own laboratory. Pyrola, mainly distributing in temperate and cold temperate regions of the northern hemisphere, has been used as sedatives and analgesics against rheumatoid arthritis and hemostatics at low prices. It is also used as a kind of tea called Lu shou cha for daily drinking in China because of its function of slowing down ageing and boosting immunity (Sun et al., 2011; Klyomi et al., 1992). Chimaphilin, a main chemical component of pyrola, not only has the antifungal activities toward S. cerevisiae, M. globosa and M. restricta, but also has the antioxidant activities through DPPH assay (Galván et al., 2008). However, the antitumor activity of chimaphilin has not been reported. The chemical structure of chimaphilin was shown in Fig. 1. Naphthalene quinonoids tend to induce acute production of ROS and alterations of intracellular redox status in cells, subsequently leading to cell death (Lin et al., 2007). Generation of ROS plays a vital role in apoptosis of hepatocellular carcinoma cells exposed to Shikonin, a naphthoquinone isolated from the Chinese herbal plant Lithospermum erythrorhizon (Gong and Li, 2011). Apoptosis induced by vitamin K3 and vitamin C alone or in combination in leukemia cells is also through the mechanism involving in oxidants stress (Bonilla-porras et al., 2011). Based on these, the cytotoxic activity of chimaphilin toward human breast cancer MCF-7 cells and the underlying mechanisms were investigated. In the present investigation, proliferation inhibition of MCF-7 cells was examined by the MTT assay. Chromatin condensation was determined by acridine orange stain assay. Apoptosis analysis, generation of ROS and disruption of mitochondrial membrane potential were conducted by flow cytometry. DNA fragmentations were determined using agarose gel electrophoresis. The expression level changes of caspase-3, caspase-9, Bcl-2, Bad and PARP proteins in MCF-7 cells treated with chimaphilin were measured by Western blotting. To the best of our knowledge, it is first reported to investigate the cytotoxic activity and underlying mechanisms of chimaphilin toward human breast cancer MCF-7 cells.
111
2. Materials and method
112
2.1. Chemicals and cell line
113 114 115 116 117 118 119 120 121 122 123 124 125 126
Chimaphilin (purity P 98%) was isolated from pyrola [Passiflora incarnata Fisch.] in our own laboratory. The isolation process was that the crude extract of pyrola extracted by ultrasound-assisted extraction was fractionated sequentially by using petroleum ether, ethyl acetate, and n-butanol, individually, in order to obtain the ethyl acetate fraction. The ethyl acetate fraction was loaded to semipreparative liquid chromatography system, and then a yellow needle crystal was obtained and identified as chimaphilin by fragment ions of ESI-MS/MS (Yao et al., 2013). The chemical structure was identified in our lab and shown in Fig. 1. A 10 mM stock solution of chimaphilin was prepared and stored at 80 °C. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), acridine orange (AO) and rhodamine 123 were obtained from Sigma–Aldrich Inc (St. Louis, MO). DCFH-DA and annexinV-FITC/propidium iodide (PI) were obtained from Beyotime Institute of Biotechnology (Beijing, China). Deionized water was used in all experiments.
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109
Fig. 1. The chemical structure of chimaphilin.
The human breast carcinoma MCF-7 cell line was purchased from Harbin Medical University, China. The cells were maintained in DMEM/HIGH Glucose (1) 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 containing 5% CO2.
127 128 129 130 131
2.2. Cell proliferation assay
132
Inhibition of cell proliferation by chimaphilin was measured by the MTT assay. MCF-7 cells were plated in 96-well culture plates at the density of 1 106 cells per well, and incubated for 24 h. Then cells were exposed to different concentration of chimaphilin for 24 h, 48 h and 72 h, respectively. MTT, which dissolved in phosphate-buffered saline at a dose of 5 mg/ml, was added. After incubation for 4 h at 37 °C, the purple formazan crystals were dissolved with 100 ll dimethyl sulfoxide and the absorbance was measured at 570 nm in an ELISA reader (Thermo Molecular Devices Co., Union City, USA). It was also measured by MTT assay in order to investigate whether chimaphilin had the cytotoxicity toward HepG-2 cells, HCT-8 cells, SMMC-7721 cells. Cytotoxicity was expressed using the IC50 value defined as the concentration of chimaphilin inhibiting cell proliferation by 50%. The cell viability ratio was calculated by the following formula: cell viability ratio (%) = ODtreated/ ODcontrol 100%.
133 134 135 136 137 138 139 140 141 142 143 144 145
2.3. Morphological changes of cell nucleus assay
146
Morphological changes of cell nucleus of MCF-7 cells exposed to chimaphilin were measured with the strain acridine orange. After incubation for 24 h in a 6-well culture plate, MCF-7 cells were treated with different concentration of chimaphilin for 24 h at 37 °C. Washed slow twice with PBS, cells were stained with 10 lg/ml AO for 8 mine at dark. The apoptosis cells were visualized using inverted fluorescence microscope (Nikon TE2000, Tokyo, Japan).
147 148 149 150 151 152
2.4. Cell apoptosis analysis
153
Apoptosis of MCF-7 cells induced by chimaphilin were detected by translocation of phosphatidylserine to the cell surface using an annexin V-FITC apoptosis detection kit (KeyGEN Biotech, China). After incubation in 6-well culture plates for 24 h, cells were exposed to different concentration of chimaphilin for 24 h at 37 °C. Washed twice with PBS, cells were resuspended in annexinV-binding buffer, incubated with annexinV-FITC/PI in dark for 10 min according to manufacture’s instructions. Subsequently the results were analyzed by flow cytometry using FloMax software.
154 155 156 157 158 159 160 161
2.5. DNA fragmentation assay
162
DNA fragmentation in MCF-7 cells exposed to chimaphilin was assayed by agarose gel electrophoresis (Wang et al., 2009). After incubation in 6-well culture plates for 24 h, cells were exposed to different concentration of chimaphilin for 24 h at 37 °C. Cells were harvested and washed twice with PBS. Total DNA was purified with a DNA isolation kit (Waston Biotechnologies Inc., Shanghai, China) according to the manufacturer’s instructions. Then DNA was separated in 1% agarose gel and visualized by ultraviolet illumination (Image Master VDS-CL, Tokyo, Japan) after staining with ethidium bromide.
163 164 165 166 167 168 169 170
2.6. Mitochondrial membrane potential disruption assay
171
Mitochondrial membrane potential disruption was estimated using the fluorescent cationic dye Rhoda mine 123. After incubation in 6-well culture plates for 24 h, cells were exposed to different concentration of chimaphilin for 24 h at 37 °C. Harvested and washed twice with PBS, cells were resuspended in fresh incubated medium containing 1.0 lM Rhoda mine 123 and incubated at 37 °C for 30 mines. Then the cells were washed twice with PBS and analyzed by means of flow cytometry.
172 173 174 175 176 177
2.7. Generation of ROS assay
178
ROS generation of MCF-7 cells induced by chimaphilin was measured using the stain of DCFH-DA by flow cytometry. After incubation in 6-well culture plates for 24 h, cells were exposed to different concentration of chimaphilin for 24 h at 37 °C. Cells were washed twice with PBS. Then cells were resuspended in fresh incubated medium containing 10 lM DCFH-DA and analyzed by the means of flow cytometry.
179 180 181 182 183 184
2.8. Protein isolation, gel electrophoresis and Western blotting
185
To investigate the expression changes of the apoptosis-related protein in MCF-7 cells exposed to chimaphilin, MCF-7 cells were exposed to different concentration of chimaphilin for 24 h at 37 °C. Then cells were harvested, washed twice with ice-cold PBS and lysed using cell lysis buffer (Beyotime Institute of Biotechnology,
186 187 188 189
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205
Beijing, China). The lysates were harvested and centrifuged at 12000 rpm at 4 °C for 5 mines. Protein concentrations of cell lysates were measured using the BCA protein assay kit (Beyotime Institute of Biotechnology, Beijing, China). Total protein samples (20 lg) were loaded on a 12% of SDS-polyactylamide gel for electrophoresis, and then 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). After incubation overnight at 4 °C with primary antibodies (anti-actin, anti-caspase-3, anticaspase-9 were mouse polyclonal antibodies; anti-Bcl-2, anti-PARP and anti-Bad were rabbit polyclonal antibodies) at a 1:1000 dilution in blocking solution (Beyotime Institute of Biotechnology, Beijing, China), membranes were incubated for 1 h at room temperature with an alkaline phosphatase peroxidase-conjugated antimouse or anti-rabbit secondary antibodies. Detection was performed by the BCIP/ NBT Alkaline Phosphates Color Development Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Bands were recorded by a digital camera (Canon, EOS350D, Tokyo, Japan).
206
2.9. Statistical analysis
207 208 209
The data were expressed as mean ± S.D. Significant differences between groups were analyzed by one-way factorial analysis of variance (ANOVA) or Student’s ttest. The results were considered statistically significant at P < 0.05.
210
3. Results
211
3.1. Chimaphilin inhibited proliferative activity of MCF-7 cells
212
221
The anti-proliferative activity of chimaphilin toward MCF-7 cells was detected by the MTT assay. As shown in Fig. 2, chimaphilin inhibited the growth of MCF-7 cells in a dose-dependent manner rather than a time-dependent manner. After treatment with chimaphilin for 72 h, 48 h and 24 h, the IC50 values were 44.10 ± 1.84 lM, 43.26 ± 2.06 lM, 43.30 ± 2.47 lM, respectively. However, the IC50 values of chimaphilin toward HepG-2 cells, HCT-116 cells, and SMMC-7721 cells for 72 h were 95.06 ± 1.03 lM, 87.64 ± 2.56 lM, and 102.56 ± 2.32 lM, respectively. Therefore, MCF-7 cell was chosen for the following test.
222
3.2. Chimaphilin induced apoptosis in MCF-7 cells
223
Chromatin condensation and morphology changes of cell nucleus are a crucial characteristic in the process of apoptosis. Chromatin condensation and morphology of cell nucleus in MCF7 cells treated with chimaphilin were examined with acridine orange stain assay. In the untreated cells, there were multiple nuclei. On the contrary, we could observe the chromatin condensation in MCF-7 cells treated with 20 lM chimaphilin for 24 h, whose fluorescence intensities were brighter. Crescent-shaped nuclei, which are one of the classic characteristics of apoptotic cells, were also found (Fig. 3). To evaluate chimaphilin-induced apoptosis in MCF-7 cells, apoptotic cells were measured using the stain of annexinV-FITC/
213 214 215 216 217 218 219 220
224 225 226 227 228 229 230 231 232 233 234
Fig. 2. Effect of chimaphilin toward MCF-7 cells as determined by the MTT assay. MCF-7 cells were treated with specified concentrations of chimaphilin for indicated time (24 h, 48 h and 72 h). The experiment was repeated three times.
3
PI after treatment with chimaphilin for 24 h. As shown in Fig. 4A and B, there were only small percentages (1.04% ± 0.93%) of cells binding annexin V-FITC in cells untreated with chimaphilin. On the contrary, the percentages of apoptotic cells were significantly increased from 5.87% ± 1.05% to 45.27% ± 1.98% and 64.93% ± 2.04% in a concentration-dependent manner after treatment with 20–40 lM chimaphilin. NAC was usually used as a free radical scavenger. When cells were treated with 10 mM NAC for 30 mines following 40 lM chimaphilin, apoptosis of cells were inhibited and the percentages of apoptotic cells were from 59.01% ± 1.46% to 29.11% ± 1.35% (Fig. 4C and D). Degradation of nuclear DNA into nucleosomal units is one of the hallmarks of apoptotic cell. It occurs in response to various apoptotic stimuli in a wide variety of cell types (Nagata, 2000). In MCF-7 cells treated with chimaphilin for 24 h, there were generations of DNA fragmentation (Fig. 5). Generation of DNA fragmentation was observed in MCF-7 cells exposed to concentrations of 40 lM chimaphilin. DNA laddering was not visible after cells were exposed to 20 and 30 lM chimaphilin. DNA fragmentation did not appear in cells untreated with chimaphilin. Taken together, the results of chromatin condensation and morphology assay, apoptosis assay and DNA fragmentation assay indicated that chimaphilin could induce apoptosis of MCF-7 cells.
235
3.3. Mitochondrial pathway was involved in chimaphilin-induced apoptosis in MCF-7 cells
258
It has been generally agreed that mitochondrial membrane potential disruption irreversibly leads to cell death involved in mitochondrial apoptosis pathway, because of release of mitochondrial apoptogenic factors and a decrease of ATP generation (Scarlett et al., 2000; Ly et al., 2003; Hu, 2003). Therefore, we investigated disruption of mitochondrial membrane potential with the stain of rhodamine 123 in MCF-7 cells treated or untreated with chimaphilin by flow cytometry. The flow cytometry assay revealed that fluorescence intensity of Rhoda mine 123 significantly decreased from 91.99% ± 1.20% to 67.64% ± 1.32%, 17.77% ± 1.98%, and 3.38% ± 2.09% in a dose-dependent manner (Fig. 6A and B). Besides, NAC could also inhibit chimaphilin-induced mitochondrial membrane potential disruption, and the percentage in cells treated with 40 lM chimaphilin was 33.92% ± 1.53% in compare with those treated with 10 mM NAC for 30 mines following 40 lM chimaphilin with the percentage of 70.98% ± 1.32%, suggesting chimaphilin induced apoptosis in MCF-7 cells involved in mitochondrial apoptosis pathway (Fig. 6C and D).
260
3.4. Generation of ROS triggered chimaphilin-induced apoptosis in MCF-7 cells
278
The generation of intracellular ROS and depletion of GSH are always associated with mitochondrial membrane potential disruption and cell apoptosis (Xiong et al., 2006; López et al., 2006). We analyzed changes of intracellular ROS in chimaphilin-treated and untreated MCF-7 cells by flow cytometry. The levels of intracellular ROS were monitored with the stain of DCFH-DA by flow cytometry. Levels changes of ROS in MCF-7 cells exposed to chimaphilin for 24 h were shown in Fig. 7A and B. Chimaphilin could induce ROS generation in MCF-7 cells and increase intracellular ROS levels in a concentration-dependent manner. After treatment with 20 lM, 30 lM and 40 lM chimaphilin, levels of intracellular ROS had a change from 44.88% ± 1.20% to 60.19% ± 1.98%, and 75.03% ± 1.87%, respectively. NAC, a free radical scavenger, blocked the chimaphilin-induced production of ROS (Fig. 7C and D). Cell counts of ROS generation were 42.04% ± 2.06% in MCF-7 cells treated with 10 mM NAC following 40 lM chimaphilin, while counts of MCF-7 cells only exposed to 40 lM chimaphilin reached to
280
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257
259
261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277
279
281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 4
W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
Fig. 3. Morphological observation of MCF-7 cells treated with chimaphilin for 24 h by the AO staining assay. (A) untreated cells; (B) treated with 20 lM chimaphilin. Cells undergoing apoptosis were indicated by white arrows indicated cells with nuclear fragmentation. The experiment was repeated three times.
Fig. 4. Chimaphilin-induced apoptosis in MCF-7 cells by annexinV-FITC/PI staining assay. The cells were treated with specific concentration of chimaphilin for 24 h. (A) Flow cytometry assay of chimaphilin-induced apoptosis cells. (a) treatment with 0 lM chimaphilin; (b) treatment with 20 lM chimaphilin; (c) treatment with 30 lM chimaphilin; (d) treatment with 40 lM chimaphilin. (B) Columns show mean values of three experiments (±S.D.). (C) NAC inhibited chimaphilin-induced apoptosis of MCF-7 cells. (a) treatment with 0 lM chimaphilin; (b) treatment with 40 lM chimaphilin; (c) treatment with 10 mM NAC following 40 lM chimaphilin; (d) treatment with 10 mM NAC. (B) Columns show mean values of three experiments (±S.D.). p < 0.05; p value compared with the control group.
297 298
55.56% ± 2.56% in compare with untreated cells (42.10% ± 1.93%) and cells only treated with NAC (43.79% ± 2.32%). The results were
proposed that chimaphilin-elicited ROS triggered chimaphilininduced apoptosis of MCF-7.
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
299 300
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
(Shi, 2002). In response to proapoptotic signals, caspase cascades are activated and function, resulting in disassembly of the cell. At the same time, apoptosis are significantly regulated by the Bcl-2 protein family, which decide whether a cell will live or die. PARP, a substrate of caspase cascades, is cleaved, ultimately leading to cell death (Gross, 1999). Fig. 8A showed that the expression levels of caspase-3 and caspase-9 were decreased after treatment with different concentration of chimaphilin for 24 h. Moreover, the remarkable cleavage of caspases-3 and caspase-9 were observed in compared with control cells. The Bcl-2 protein levels decreased in MCF-7 cells, while the Bad protein levels increased. Moreover, the cleavage of PARP was also observed. Western blotting showed that the intact 116 kDa moieties of PARP were decreased and that the cleaved forms (89 kDa) were increased in MCF-7 cells treated with 40 lM chimaphilin. In contrast, MCF-7 cells were treated with 10 mM NAC for 30 mines following 40 lM chimaphilin. Western blotting showed that NAC could inhibit the decrease of Bcl-2, the increase of Bad, the cleavage of caspase-3, caspase-9 and PARP in apoptotic cells induced by chimaphilin (Fig. 8B). In a word, chimaphilin could induce apoptosis involving in ROS-mediated mitoQ2 chondrial pathway in MCF-7 cells. (see Fig. 9).
305
3.5. Chimaphilin activated caspases and the execution phase of apoptosis
4. Discussion
326
The caspase-cascade system plays crucial roles in cell apoptosis, and it is reported that caspases are the executors of apoptosis
Quinones are one of the most clinical anticancer drugs in the world, which have different active function. In apoptotic cells,
327
Fig. 5. DNA fragmentation in MCF-7 cells treated with chimaphilin. Lane 1, treatment with 0 lM chimaphilin; lane 2, treatment with 20 lM chimaphilin; lane 3, treatment with 30 lM chimaphilin; lane 4, treatment with 40 lM chimaphilin; lane 5, DNA size marker. The experiment was repeated three times.
301 302 303 304
5
Fig. 6. Mitochondrial membrane potential disruption in MCF-7 cells treated with chimaphilin. Cells were treated with different concentration of chimaphilin for 24 h. (A) Flow cytometry assay of chimaphilin-induced mitochondrial membrane potential disruption of cells. (a) treatment with 0 lM chimaphilin; (b), treatment with 20 lM chimaphilin; (c) treatment with 30 lM chimaphilin; (d) treatment with 40 lM chimaphilin. (B) Columns show mean values of three experiments (±S.D.). (C) NAC inhibited chimaphilin-induced mitochondrial membrane potential disruption of cells. (a) treatment with 0 lM chimaphilin; (b) treatment with 40 lM chimaphilin; (c) treatment with 10 mM NAC following 40 lM chimaphilin; (d) treatment with 10 mM NAC. (D) Columns show mean values of three experiments (±S.D.). p < 0.05; p value compared with the control group.
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325
328
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 6
W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
Fig. 7. ROS generation in MCF-7 cells treated with chimaphilin. Cells were treated with different concentration of chimaphilin for 24 h. (A) Flow cytometry assay of chimaphilin-induced ROS generation of cells. (a) treatment with 0 lM chimaphilin; (b) treatment with 20 lM chimaphilin; (c) treatment with 30 lM chimaphilin; (d) treatment with 40 lM chimaphilin. (B) Columns show mean values of three experiments (±S.D.). (C) NAC inhibited chimaphilin-induced ROS generation of cells. (a) treatment with 0 lM chimaphilin; (b) treatment with 40 lM chimaphilin; (c) treatment with 10 mM NAC following 40 lM chimaphilin; (d) treatment with 10 mM NAC. (D) Columns show mean values of three experiments (±S.D.). p < 0.05; p value compared with the control group.
Fig. 8. Effect of chimaphilin on apoptosis-related proteins involved in mitochondria/caspase-indepented signaling pathway in MCF-7 cells. A The expression level of caspase3, caspase-9, PARP, Bcl-2 and bad, was detected by western blotting in MCF-7 cells treated with chimaphilin for 24 h. B The cells was pretreated for 30 min by N-acety-Lcystein before treatment with chimaphilin. Then the expression levels of caspase-3, caspase-9, PARP, Bcl-2 and Bad were detected.
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
7
Fig. 9. Signaling pathway of apoptosis induced by chimaphilin in MCF-7 cells.
329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365
naphthoquinone derivatives generally lead to generation of reactive oxygen species through redox cycling, consisting with inhibiting NAD(P)H: quinone oxidoreductase. The naphthoquinone derivatives have the chemical structure of quinone with gaining electrons (Criddle et al., 2006). Menadione, a naphthoquinone, triggers apoptosis of mouse myogenic C2C12 cells through generation of reactive oxygen species, oxidative stress, iron-dependent lipid peroxidation, and glutathione depletion (Chiou et al., 2003). In U87MG glioma cells, shikonin also induces the generation of ROS, depletion of GSH, disruption of mitochondrial membrane potential, upregulation of p53, and cleavage of PARP (poly(ADP-ribose) polymerase). The changes contribute U87MG glioma cells to apoptosis (Chen et al., 2012). Chimaphilin, isolated from pyrola [Passiflora incarnata Fisch.], is also a 1,4-naphthoquinone derivative. However, there have been no reports about the antitumor mechanism of chimaphilin. In this study, we first investigated the anticancer activity of chimaphilin toward human breast cancer MCF-7 cells. In addition, we presumed that chimaphilin could trigger apoptosis of human breast cancer MCF-7 cells through the mechanism of a ROS-mediated mitochondrial pathway. In the therapies of carcinoma, apoptosis is a main approach. Apoptosis plays a crucial role in the cellular progress of proliferation, differentiation, senescence, and death. The MTT assay was carried out to evaluate the cytotoxicity of chimaphilin toward human breast cancer MCF-7 cells. Chimaphilin inhibited the growth of MCF-7 cells in a dose- not time-dependent manner (Fig. 2), but it could not inhibit the proliferation of HepG-2 cells, HCT-116 cells and SMMC-7721 cells. As known in apoptotic cells, phosphatidylserine is translocated from the inner to the outer leaflet of the plasma membrane. The stain of annexinV-FITC/PI was used to explore whether apoptosis occurred in MCF-7 cells exposed to chimaphilin. In compared with cells without treatment of chimaphilin, fluorescence intensities of cells were increased at the concentration of 30 and 40 lM chimaphilin (Fig. 4A and B). Nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation are significant characteristic of apoptosis. Chromatin condensation was observed in MCF-7 cells treated with
20 lM chimaphilin (Fig. 3). Moreover, DNA Ladder was also observed by agarose gel electrophoresis assay (Fig. 5). Consequently, it was demonstrated that chimaphilin could initiate apoptosis of human breast carcinoma MCF-7 cells. As a major compartment of energy metabolism, mitochondrial plays a pivotal role in the progress of caspase-denpendent apoptosis. When antitumor reagents stimulate cancer cells, pro-apoptotic signals regulate and accumulate the expression of pro- and antiapoptosis Bcl-2 family proteins in the cytoplasm. Then these proteins converge on mitochondria to change mitochondrial outer membrane permeabilization, which results in the release of cytochrome c, activation of caspase cascades, and apoptosis (Douglas and Kroemer, 2004; Douglas and John, 1998). In MCF-7 cells, chimaphilin caused intracellular high Bad/Bcl-2 ratio (Fig. 8A and B), which contributed to reduce mitochondrial membrane potential (Fig. 6A and B) and evoke mitochondrial outer membrane permeabilization, resulting in the release of cytochrome c from mitochondria to cytoplasm matrix. Once cytochrome c binds Apaf-1 and procaspase-9, procaspase-9 is cleaved and activates procaspase-3 (Jiang and Wang, 2004). PARP, known as a poly (ADP-ribose) synthetase and poly (ADP-ribose) transferase, catalyzes the transfer of the ADP-ribose moiety from the respiratory co-enzyme NAD+ to a limited number of acceptor proteins involved in chromatin architectures when cells are stimulated by DNA strand-breaks (Van wijk and Hageman, 2005; Huber et al., 2004). PARP is generally cleaved as a substrate by caspase-3 in apoptosis (Zhang et al., 2005). As shown in Fig. 8A and B, chimaphilin activated caspase-9, caspase-3 and cleavage of PARP in MCF-7 cells. Accordingly, we found that MCF-7 cells treated with chimaphilin were subjected to apoptosis involving in mitochondria pathway. It has been reported by many authors that ROS can induce apoptosis in many different cell systems (Simon et al., 2000). Hydrogen peroxide-induced apoptosis is required for the release of mitochondria-derived ROS and activation of caspase-3 (Dumont et al., 1999). ROS elicited by menadione via redox can induce apoptosis of murine pancreatic acinar cells (Criddle et al., 2006). Cajanol, a novel isoflavanone from pigeonpea, also triggers
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 8
W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
416
apoptosis through a ROS-mediated mitochondrial pathway (Meng et al., 2010). ROS from mitochondrial may oxidize membrane proteins of mitochondrial, change mitochondrial outer membrane permeabilization, and lead to disruption of mitochondrial membrane potential, which contribute to the release of cytochrome c and apoptosis (Petrosillo et al., 2003). Chimaphilin could cause ROS generation and lead to apoptosis of MCF-7 cells in a concentration-dependent manner. The intracellular ROS increase was consistent to apoptosis induced by chimaphilin in MCF-7 cells (Fig. 7A and B). NAC, a scavenging reagent of ROS, could prevent chimaphilin-induced disruption of mitochondrial membrane potential and apoptosis in MCF-7 cells (Fig. 6C, D and Fig. 4C, D). Therefore, our results suggested that the main cause of chimaphilin-induced apoptosis was ROS.
417
5. Conclusion
418
425
The present study demonstrated that chimaphilin could induce apoptosis of MCF-7 cells through a ROS-regulated mitochondrial pathway. Generation of ROS was one cause of apoptosis induced by chimaphilin. Further research is needed to pinpoint other pivotal signaling pathways induced by chimaphilin toward human breast cancer MCF-7 cells. Our results might provide the basis data for future clinical application of chimaphilin treatment in breast cancer.
426
Conflict of Interest
403 404 405 406 407 408 409 410 411 412 413 414 415
419 420 421 422 423 424
427
The authors declare that there are no conflicts of interest.
428
Transparency Document
429
The Transparency document associated with this article can be found in the online version.
430 431
432
Acknowledgements
433
438
The authors gratefully acknowledge the financial supports by, Program for Agricultural Science and Technology Achievements Transformation Fund Program (2012GB23600641), Program for Importation of International Advanced Forestry Science and Technology, National Forestry Bureau (2012-4-06), Special Fund of National Natural Science Foundation of China (31270618).
439
References
440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460
Apel, K.H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399. Bonilla-porras, A.R., Jimenez-del-rio, M., Velez-pardo, C., 2011. Vitamin K3 and vitamin C alone or in combination induced apoptosis in leukemia cells by a similar oxidative stress signalling mechanism. Cancer Cell Int. 11, 1–11. Chen, C.H., Lin, M.L., Ong, P.L., Yang, J.T., 2012. Novel multiple apoptotic mechanism of shikonin in human glioma cells. Ann. Surg. Oncol. 19, 3097–3106. Chiou, T.J., Chu, S.T., Tzeng, W.F., 2003. Protection of cells from menadione-induced apoptosis by inhibition of lipid peroxidation. Toxicology 191, 77–88. Criddle, D.N., Gillies, S., Baumgartner-wilson, H.K., Jaffar, M., Chinje, E.C., Passmore, S., Chvanov, M., Barrow, S., Gerasimenko, O.V., Tepikin, A.V., Sutton, R., Petersen, O.H., 2006. Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells. J. Biol. Chem. 281, 40485–40492. Crompton, M., Barksby, E., Johnson, N., Capano, M., 2002. Mitochondrial intermembrane junctional complexes and their involvement in cell death. Biochimie 84, 143–152. Douglas, R.G., John, C.R., 1998. Mitochondria and apoptosis. Science 281, 1309– 1312. Douglas, R.G., Kroemer, G., 2004. The pathophysiology of mitochondrial cell death. Science 305, 626–629.
434 435 436 437
Dumont, A., Hehner, S.P., Hofmann, T.G., et al., 1999. Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB. Oncogene 18, 747– 757. Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35 (4), 495–516. Galván, I.J., Mir-rashed, N., Jessulat, M., Atanya, M., Golshani, A., Durst, T., Petit, P., Amiguet, V.T., Boekhout, T., Summerbell, R., Cruz, I., Arnason, J.T., Smith, M.L., 2008. Antifungal and antioxidant activities of the phytomedicine pipsissewa, Chimaphila umbellata. Phytochemistry 69, 738–746. Gong, K., Li, W., 2011. Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen species: a potential new treatment for hepatocellular carcinoma. Free Radical Biol. Med. 51, 2259–2271. Gross, A.J., 1999. BCL-2 family members and the mitochondria in apoptosis. Genes 13, 1899–1911. Hu, W.J.J., 2003. Anticancer therapy targeting the apoptotic pathway. Lancet Oncol. 4, 721–729. Huber, A., Bai, P., De murcia, J.M., et al., 2004. PARP-1, PARP-2 and ATM in the DNA damage response: functional synergy in mouse development. DNA Repair 3, 1103–1108. Jiang, X., Wang, X., 2004. Cytochrome C-mediated apoptosis. Annu. Rev. Biochem. 73, 87–106. Klyomi, K., Katsuya, T., Kiyohisa, U., Hisato, K., Tsutomu, S., Hiroshi, N., 1992. Platelet aggregation inhibitors and inotropic constituents in Pyrolae herba. Chem. Pharm. Bull. 40, 2083–2087. Lin, C.H., Huang, C.C., Wang, T.W., Wang, Y.J., Lin, P.H., 2007. Disparity in the induction of glutathione depletion, ROS formation, poly(ADP-ribose) polymerase-1 activation, and apoptosis by quinonoid derivatives of naphthalene in human cultured cells. Chem. Biol. Interact. 165, 200–210. López, E., Arce, C., Oset-gasque, M.J., Cañadas, S., González, M.P., 2006. Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture. Free Radical Biol. Med. 40, 940–951. Ly, J.D., Grubb, D.R., Lawen, A., 2003. The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis: Int. J. Programmed 8, 115– 128. Meng, L., Xia, L., Zu, Y., Fu, Y.J., Su, Z., Yao, L.P., Efferth, T., 2010. Cajanol, a novel anticancer agent from Pigeonpea [Cajanus cajan (L.) Millsp.] roots, induces apoptosis in human breast cancer cells through a ROS-mediated mitochondrial pathway. Chem. Biol. Interact. 188, 151–160. Nagata, S., 2000. Apoptotic DNA fragmentation. Exp. Cell Res. 256, 12–18. Natarajan, N., Thamaraiselvan, R., Lingaiah, H., et al., 2011. Effect of flavonone hesperidin on the apoptosis of human mammary carcinoma cell line MCF-7. Biomed. Prev. Nutr. 1, 207–215. Noori, S., Hassan, Z.M., 2012. Tehranolide inhibits proliferation of MCF-7 human breast cancer cells by inducing G0/G1 arrest and apoptosis. Free Radical Biol. Med. 52, 1987–1999. Peter, M.E., Heufelder, A.E., Hengartner, M.O., 1997. Advances in apoptosis research. Proc. Natl. Acad. Sci. 94 (24), 12736–12737, Proceedings of the National Academy of Sciences 94, 11273–12736. Petrosillo, G., Ruggiero, F.M., Paradies, G., 2003. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria. FASEB J. 17, 2202–2208. Scarlett, J.L., Sheard, P.W., Hughes, G., Ledgerwood, E.C., Ku, H.H., Murphy, M.P., 2000. Changes in mitochondrial membrane potential during staurosporineinduced apoptosis in Jurkat cells. FEBS Lett. 475, 267–272. Shi, Y., 2002. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell 9, 459–470. Simon, H.U., Haj-yehia, A., Levi-schaffer, F., 2000. Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 5, 415–418. Sun, Y.A., 1999. Menadione-induced apoptosis and the degradation of lamin-like proteins in tobacco protoplasts. Cell. Mol. Life Sci. CMLS 55, 310–316. Sun, C., Chen, Z., Wang, W., Li, Y., Dong, Y., Wang, L., 2011. Research on the development and utilization of Pyrola resources. Northern Horticulture 1, 220– 222. Van wijk, S.J., Hageman, G.J., 2005. Poly (ADP-ribose) polymerase-1 mediated caspase-independent cell death after ischemia/reperfusion. Free Radical Biol. Med. 39, 81–90. Vinatier, D., Dufour, P., Subtil, D., 1996. Apoptosis: a programmed cell death involved in ovarian and uterine physiology. Eur. J. Obstet. Gynecol. Reprod. Biol. 67, 85–102. Wang, H., Xu, Y., Yan, J., Zhao, X., Sun, X., Zhang, Y., Guo, J., Zhu, C., 2009. Acteoside protects human neuroblastoma SH-SY5Y cells against beta-amyloid-induced cell injury. Brain Res. 1283, 139–147. Xiong, Y., Liu, X., Lee, C.P., Chua, B.H., Ho, Y.S., 2006. Attenuation of doxorubicininduced contractile and mitochondrial dysfunction in mouse heart by cellular glutathione peroxidase. Free Radical Biol. Med. 41, 46–55. Yao, X.H., Zhang, D.Y., Zu, Y.G., Fu, Y.J., Luo, M., Gu, C.B., Efferth, T., 2013. Free radical scavenging capability, antioxidant activity and chemical constituents of Pyrola incarnata Fisch. leaves. Ind. Crops Prod. 49, 247–255. Zhang, Y., Zhang, X., Park, T.S., et al., 2005. Cerebral endothelial cell apoptosis after ischemia–reperfusion: role of PARP activation and AIF translocation. J. Cerebral Blood Flow Metab. 25, 868–877.
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544
No. of Pages 8, Model 5G
6 May 2014 Food and Chemical Toxicology xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox 5 6
Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway
3 4 7 8 9 10 11 12 1 2 4 6 15 16 17 18 19 20 21 22 23 24 25
Q1
Wei-Dong Ma a,1, Yong-Peng Zou b,1, Peng Wang a, Xiao-Hui Yao a, Yao Sun a, Ming-Hui Duan a, Yu-Jie Fu a,⇑, Bo Yu b,⇑ a b
Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China The 2nd Affiliated Hospital of Harbin Medical University, Harbin 150001, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 17 October 2013 Accepted 8 April 2014 Available online xxxx
Chimaphilin, 2,7-dimethyl-1,4-naphthoquinone, is extracted from pyrola [Passiflora incarnata Fisch.]. In this study, the anticancer activity and underlying mechanisms of chimaphilin toward human breast cancer MCF-7 cells are firstly investigated. Chimaphilin could inhibit the viability of MCF-7 cells in a concentration-dependent manner, and the IC50 value was 43.30 lM for 24 h. Chimaphilin markedly induced apoptosis through the investigation of characteristic apoptotic morphological changes, nuclear DNA fragmentation, annexin V-FITC/propidium iodide (PI) double staining. Flow cytometry assay revealed that chimaphilin triggered a significant generation of ROS and disruption of mitochondrial membrane potential. Additionally, western blotting assay showed that chimaphilin suppressed Bcl-2 level and enhanced Bad level, then activated caspase-9 and caspase-3, and further activated the poly ADP-ribose polymerase (PARP), finally induced cell apoptosis involving the mitochondrial pathway. Furthermore, free radical scavengers N-acetyl-L-cysteine (NAC) pretreatment test testified that chimaphilin could increase the generation of ROS, then induce cell apoptosis. In general, the present results demonstrated that chimaphilin induced apoptosis in human breast cancer MCF-7 cells via a ROS-mediated mitochondrial pathway. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Chimaphilin Apoptosis Reactive oxygen species Mitochondrial MCF-7
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
44 45
1. Introduction
46
Cancer is one of the major diseases leading to deaths in the world (Natarajan et al., 2011). In the developed world, breast cancer is one of the most common types of cancers among women, which accounts for 15–20% of all cancer deaths in the mid-1990s (Noori and Hassan, 2012). Chemotherapy and radiotherapy are
47 48 49 50
0
0
Abbreviations: AO, acridine orange; DCFH-DA, 2 ,7 -dichlorofluorescin diacetate; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl; MCF-7 cells, human breast cancer cell line; MMP, mitochondrial membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; NAC, N-acety-L-cystein; ROS, reactive oxygen species; PI, propidium iodide. ⇑ Corresponding authors. Address: State Engineering Laboratory of Bio-Resource Eco-Utilization, Northeast Forestry University, Hexing Road, Harbin 150040, PR China. Tel./fax: +86 451 82190535 (Y. Fu). Address: Key Laboratory of Myocardial Ischemia, Ministry of Education, The 2nd Affiliated Hospital of Harbin Medical University, Xuefu Road, Harbin 150001, PR China. Tel./fax: +86 451 86297220 (B. Yu). E-mail addresses: [email protected] (Y.-J. Fu), [email protected] (B. Yu).
1
main therapeutic method of cancer at present. However, chemotherapy and radiotherapy were generally expensive for patients. Additionally, chemotherapy generally leads to drug resistance and severe side effects for patients. Therefore, it is necessary to develop high efficient natural chemotherapeutic agents with low cost, low drug resistance and side effects. Apoptosis is a process of genetically programmed cell death that consists of a cascade of molecular events in stimulated cells (Vinatier et al., 1996). Apoptosis is triggered by biological and physical signals, such as chemical reagents, radiation, free radicals, and infection of virus et al. (Sun, 1999; Peter et al., 1997). The mechanism of apoptosis is involved in an energy-dependent cascade of molecular events. To date, the extrinsic death receptor pathway and the intrinsic mitochondrial pathway are two main apoptotic pathways (Elmore, 2007). In apoptotic cells, over-expression of intracellular reactive oxygen species (ROS) is a main cause of leading to DNA and protein damage. ROS, which consists of some radical species and non-radicals hydrogen peroxide, plays a key role in metabolism, oxidative stress, signal transduction, and apoptosis in cells (Apel, 2004; Simon et al., 2000). Generation of ROS can directly activate the mitochondrial permeability transition and
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.fct.2014.04.014 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 2
W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
110
result in loss of mitochondrial membrane potential (MMP) and the membrane integrity (Crompton et al., 2002). Subsequently, cytochrome C is released from mitochondria into the cytosol and activates the caspase cascades contributing to apoptosis of cells. Chimaphilin, 2,7-dimethyl-1,4-naphthoquinone, was isolated from pyrola [Passiflora incarnata Fisch.] in our own laboratory. Pyrola, mainly distributing in temperate and cold temperate regions of the northern hemisphere, has been used as sedatives and analgesics against rheumatoid arthritis and hemostatics at low prices. It is also used as a kind of tea called Lu shou cha for daily drinking in China because of its function of slowing down ageing and boosting immunity (Sun et al., 2011; Klyomi et al., 1992). Chimaphilin, a main chemical component of pyrola, not only has the antifungal activities toward S. cerevisiae, M. globosa and M. restricta, but also has the antioxidant activities through DPPH assay (Galván et al., 2008). However, the antitumor activity of chimaphilin has not been reported. The chemical structure of chimaphilin was shown in Fig. 1. Naphthalene quinonoids tend to induce acute production of ROS and alterations of intracellular redox status in cells, subsequently leading to cell death (Lin et al., 2007). Generation of ROS plays a vital role in apoptosis of hepatocellular carcinoma cells exposed to Shikonin, a naphthoquinone isolated from the Chinese herbal plant Lithospermum erythrorhizon (Gong and Li, 2011). Apoptosis induced by vitamin K3 and vitamin C alone or in combination in leukemia cells is also through the mechanism involving in oxidants stress (Bonilla-porras et al., 2011). Based on these, the cytotoxic activity of chimaphilin toward human breast cancer MCF-7 cells and the underlying mechanisms were investigated. In the present investigation, proliferation inhibition of MCF-7 cells was examined by the MTT assay. Chromatin condensation was determined by acridine orange stain assay. Apoptosis analysis, generation of ROS and disruption of mitochondrial membrane potential were conducted by flow cytometry. DNA fragmentations were determined using agarose gel electrophoresis. The expression level changes of caspase-3, caspase-9, Bcl-2, Bad and PARP proteins in MCF-7 cells treated with chimaphilin were measured by Western blotting. To the best of our knowledge, it is first reported to investigate the cytotoxic activity and underlying mechanisms of chimaphilin toward human breast cancer MCF-7 cells.
111
2. Materials and method
112
2.1. Chemicals and cell line
113 114 115 116 117 118 119 120 121 122 123 124 125 126
Chimaphilin (purity P 98%) was isolated from pyrola [Passiflora incarnata Fisch.] in our own laboratory. The isolation process was that the crude extract of pyrola extracted by ultrasound-assisted extraction was fractionated sequentially by using petroleum ether, ethyl acetate, and n-butanol, individually, in order to obtain the ethyl acetate fraction. The ethyl acetate fraction was loaded to semipreparative liquid chromatography system, and then a yellow needle crystal was obtained and identified as chimaphilin by fragment ions of ESI-MS/MS (Yao et al., 2013). The chemical structure was identified in our lab and shown in Fig. 1. A 10 mM stock solution of chimaphilin was prepared and stored at 80 °C. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), acridine orange (AO) and rhodamine 123 were obtained from Sigma–Aldrich Inc (St. Louis, MO). DCFH-DA and annexinV-FITC/propidium iodide (PI) were obtained from Beyotime Institute of Biotechnology (Beijing, China). Deionized water was used in all experiments.
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109
Fig. 1. The chemical structure of chimaphilin.
The human breast carcinoma MCF-7 cell line was purchased from Harbin Medical University, China. The cells were maintained in DMEM/HIGH Glucose (1) 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 containing 5% CO2.
127 128 129 130 131
2.2. Cell proliferation assay
132
Inhibition of cell proliferation by chimaphilin was measured by the MTT assay. MCF-7 cells were plated in 96-well culture plates at the density of 1 106 cells per well, and incubated for 24 h. Then cells were exposed to different concentration of chimaphilin for 24 h, 48 h and 72 h, respectively. MTT, which dissolved in phosphate-buffered saline at a dose of 5 mg/ml, was added. After incubation for 4 h at 37 °C, the purple formazan crystals were dissolved with 100 ll dimethyl sulfoxide and the absorbance was measured at 570 nm in an ELISA reader (Thermo Molecular Devices Co., Union City, USA). It was also measured by MTT assay in order to investigate whether chimaphilin had the cytotoxicity toward HepG-2 cells, HCT-8 cells, SMMC-7721 cells. Cytotoxicity was expressed using the IC50 value defined as the concentration of chimaphilin inhibiting cell proliferation by 50%. The cell viability ratio was calculated by the following formula: cell viability ratio (%) = ODtreated/ ODcontrol 100%.
133 134 135 136 137 138 139 140 141 142 143 144 145
2.3. Morphological changes of cell nucleus assay
146
Morphological changes of cell nucleus of MCF-7 cells exposed to chimaphilin were measured with the strain acridine orange. After incubation for 24 h in a 6-well culture plate, MCF-7 cells were treated with different concentration of chimaphilin for 24 h at 37 °C. Washed slow twice with PBS, cells were stained with 10 lg/ml AO for 8 mine at dark. The apoptosis cells were visualized using inverted fluorescence microscope (Nikon TE2000, Tokyo, Japan).
147 148 149 150 151 152
2.4. Cell apoptosis analysis
153
Apoptosis of MCF-7 cells induced by chimaphilin were detected by translocation of phosphatidylserine to the cell surface using an annexin V-FITC apoptosis detection kit (KeyGEN Biotech, China). After incubation in 6-well culture plates for 24 h, cells were exposed to different concentration of chimaphilin for 24 h at 37 °C. Washed twice with PBS, cells were resuspended in annexinV-binding buffer, incubated with annexinV-FITC/PI in dark for 10 min according to manufacture’s instructions. Subsequently the results were analyzed by flow cytometry using FloMax software.
154 155 156 157 158 159 160 161
2.5. DNA fragmentation assay
162
DNA fragmentation in MCF-7 cells exposed to chimaphilin was assayed by agarose gel electrophoresis (Wang et al., 2009). After incubation in 6-well culture plates for 24 h, cells were exposed to different concentration of chimaphilin for 24 h at 37 °C. Cells were harvested and washed twice with PBS. Total DNA was purified with a DNA isolation kit (Waston Biotechnologies Inc., Shanghai, China) according to the manufacturer’s instructions. Then DNA was separated in 1% agarose gel and visualized by ultraviolet illumination (Image Master VDS-CL, Tokyo, Japan) after staining with ethidium bromide.
163 164 165 166 167 168 169 170
2.6. Mitochondrial membrane potential disruption assay
171
Mitochondrial membrane potential disruption was estimated using the fluorescent cationic dye Rhoda mine 123. After incubation in 6-well culture plates for 24 h, cells were exposed to different concentration of chimaphilin for 24 h at 37 °C. Harvested and washed twice with PBS, cells were resuspended in fresh incubated medium containing 1.0 lM Rhoda mine 123 and incubated at 37 °C for 30 mines. Then the cells were washed twice with PBS and analyzed by means of flow cytometry.
172 173 174 175 176 177
2.7. Generation of ROS assay
178
ROS generation of MCF-7 cells induced by chimaphilin was measured using the stain of DCFH-DA by flow cytometry. After incubation in 6-well culture plates for 24 h, cells were exposed to different concentration of chimaphilin for 24 h at 37 °C. Cells were washed twice with PBS. Then cells were resuspended in fresh incubated medium containing 10 lM DCFH-DA and analyzed by the means of flow cytometry.
179 180 181 182 183 184
2.8. Protein isolation, gel electrophoresis and Western blotting
185
To investigate the expression changes of the apoptosis-related protein in MCF-7 cells exposed to chimaphilin, MCF-7 cells were exposed to different concentration of chimaphilin for 24 h at 37 °C. Then cells were harvested, washed twice with ice-cold PBS and lysed using cell lysis buffer (Beyotime Institute of Biotechnology,
186 187 188 189
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205
Beijing, China). The lysates were harvested and centrifuged at 12000 rpm at 4 °C for 5 mines. Protein concentrations of cell lysates were measured using the BCA protein assay kit (Beyotime Institute of Biotechnology, Beijing, China). Total protein samples (20 lg) were loaded on a 12% of SDS-polyactylamide gel for electrophoresis, and then 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). After incubation overnight at 4 °C with primary antibodies (anti-actin, anti-caspase-3, anticaspase-9 were mouse polyclonal antibodies; anti-Bcl-2, anti-PARP and anti-Bad were rabbit polyclonal antibodies) at a 1:1000 dilution in blocking solution (Beyotime Institute of Biotechnology, Beijing, China), membranes were incubated for 1 h at room temperature with an alkaline phosphatase peroxidase-conjugated antimouse or anti-rabbit secondary antibodies. Detection was performed by the BCIP/ NBT Alkaline Phosphates Color Development Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Bands were recorded by a digital camera (Canon, EOS350D, Tokyo, Japan).
206
2.9. Statistical analysis
207 208 209
The data were expressed as mean ± S.D. Significant differences between groups were analyzed by one-way factorial analysis of variance (ANOVA) or Student’s ttest. The results were considered statistically significant at P < 0.05.
210
3. Results
211
3.1. Chimaphilin inhibited proliferative activity of MCF-7 cells
212
221
The anti-proliferative activity of chimaphilin toward MCF-7 cells was detected by the MTT assay. As shown in Fig. 2, chimaphilin inhibited the growth of MCF-7 cells in a dose-dependent manner rather than a time-dependent manner. After treatment with chimaphilin for 72 h, 48 h and 24 h, the IC50 values were 44.10 ± 1.84 lM, 43.26 ± 2.06 lM, 43.30 ± 2.47 lM, respectively. However, the IC50 values of chimaphilin toward HepG-2 cells, HCT-116 cells, and SMMC-7721 cells for 72 h were 95.06 ± 1.03 lM, 87.64 ± 2.56 lM, and 102.56 ± 2.32 lM, respectively. Therefore, MCF-7 cell was chosen for the following test.
222
3.2. Chimaphilin induced apoptosis in MCF-7 cells
223
Chromatin condensation and morphology changes of cell nucleus are a crucial characteristic in the process of apoptosis. Chromatin condensation and morphology of cell nucleus in MCF7 cells treated with chimaphilin were examined with acridine orange stain assay. In the untreated cells, there were multiple nuclei. On the contrary, we could observe the chromatin condensation in MCF-7 cells treated with 20 lM chimaphilin for 24 h, whose fluorescence intensities were brighter. Crescent-shaped nuclei, which are one of the classic characteristics of apoptotic cells, were also found (Fig. 3). To evaluate chimaphilin-induced apoptosis in MCF-7 cells, apoptotic cells were measured using the stain of annexinV-FITC/
213 214 215 216 217 218 219 220
224 225 226 227 228 229 230 231 232 233 234
Fig. 2. Effect of chimaphilin toward MCF-7 cells as determined by the MTT assay. MCF-7 cells were treated with specified concentrations of chimaphilin for indicated time (24 h, 48 h and 72 h). The experiment was repeated three times.
3
PI after treatment with chimaphilin for 24 h. As shown in Fig. 4A and B, there were only small percentages (1.04% ± 0.93%) of cells binding annexin V-FITC in cells untreated with chimaphilin. On the contrary, the percentages of apoptotic cells were significantly increased from 5.87% ± 1.05% to 45.27% ± 1.98% and 64.93% ± 2.04% in a concentration-dependent manner after treatment with 20–40 lM chimaphilin. NAC was usually used as a free radical scavenger. When cells were treated with 10 mM NAC for 30 mines following 40 lM chimaphilin, apoptosis of cells were inhibited and the percentages of apoptotic cells were from 59.01% ± 1.46% to 29.11% ± 1.35% (Fig. 4C and D). Degradation of nuclear DNA into nucleosomal units is one of the hallmarks of apoptotic cell. It occurs in response to various apoptotic stimuli in a wide variety of cell types (Nagata, 2000). In MCF-7 cells treated with chimaphilin for 24 h, there were generations of DNA fragmentation (Fig. 5). Generation of DNA fragmentation was observed in MCF-7 cells exposed to concentrations of 40 lM chimaphilin. DNA laddering was not visible after cells were exposed to 20 and 30 lM chimaphilin. DNA fragmentation did not appear in cells untreated with chimaphilin. Taken together, the results of chromatin condensation and morphology assay, apoptosis assay and DNA fragmentation assay indicated that chimaphilin could induce apoptosis of MCF-7 cells.
235
3.3. Mitochondrial pathway was involved in chimaphilin-induced apoptosis in MCF-7 cells
258
It has been generally agreed that mitochondrial membrane potential disruption irreversibly leads to cell death involved in mitochondrial apoptosis pathway, because of release of mitochondrial apoptogenic factors and a decrease of ATP generation (Scarlett et al., 2000; Ly et al., 2003; Hu, 2003). Therefore, we investigated disruption of mitochondrial membrane potential with the stain of rhodamine 123 in MCF-7 cells treated or untreated with chimaphilin by flow cytometry. The flow cytometry assay revealed that fluorescence intensity of Rhoda mine 123 significantly decreased from 91.99% ± 1.20% to 67.64% ± 1.32%, 17.77% ± 1.98%, and 3.38% ± 2.09% in a dose-dependent manner (Fig. 6A and B). Besides, NAC could also inhibit chimaphilin-induced mitochondrial membrane potential disruption, and the percentage in cells treated with 40 lM chimaphilin was 33.92% ± 1.53% in compare with those treated with 10 mM NAC for 30 mines following 40 lM chimaphilin with the percentage of 70.98% ± 1.32%, suggesting chimaphilin induced apoptosis in MCF-7 cells involved in mitochondrial apoptosis pathway (Fig. 6C and D).
260
3.4. Generation of ROS triggered chimaphilin-induced apoptosis in MCF-7 cells
278
The generation of intracellular ROS and depletion of GSH are always associated with mitochondrial membrane potential disruption and cell apoptosis (Xiong et al., 2006; López et al., 2006). We analyzed changes of intracellular ROS in chimaphilin-treated and untreated MCF-7 cells by flow cytometry. The levels of intracellular ROS were monitored with the stain of DCFH-DA by flow cytometry. Levels changes of ROS in MCF-7 cells exposed to chimaphilin for 24 h were shown in Fig. 7A and B. Chimaphilin could induce ROS generation in MCF-7 cells and increase intracellular ROS levels in a concentration-dependent manner. After treatment with 20 lM, 30 lM and 40 lM chimaphilin, levels of intracellular ROS had a change from 44.88% ± 1.20% to 60.19% ± 1.98%, and 75.03% ± 1.87%, respectively. NAC, a free radical scavenger, blocked the chimaphilin-induced production of ROS (Fig. 7C and D). Cell counts of ROS generation were 42.04% ± 2.06% in MCF-7 cells treated with 10 mM NAC following 40 lM chimaphilin, while counts of MCF-7 cells only exposed to 40 lM chimaphilin reached to
280
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257
259
261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277
279
281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 4
W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
Fig. 3. Morphological observation of MCF-7 cells treated with chimaphilin for 24 h by the AO staining assay. (A) untreated cells; (B) treated with 20 lM chimaphilin. Cells undergoing apoptosis were indicated by white arrows indicated cells with nuclear fragmentation. The experiment was repeated three times.
Fig. 4. Chimaphilin-induced apoptosis in MCF-7 cells by annexinV-FITC/PI staining assay. The cells were treated with specific concentration of chimaphilin for 24 h. (A) Flow cytometry assay of chimaphilin-induced apoptosis cells. (a) treatment with 0 lM chimaphilin; (b) treatment with 20 lM chimaphilin; (c) treatment with 30 lM chimaphilin; (d) treatment with 40 lM chimaphilin. (B) Columns show mean values of three experiments (±S.D.). (C) NAC inhibited chimaphilin-induced apoptosis of MCF-7 cells. (a) treatment with 0 lM chimaphilin; (b) treatment with 40 lM chimaphilin; (c) treatment with 10 mM NAC following 40 lM chimaphilin; (d) treatment with 10 mM NAC. (B) Columns show mean values of three experiments (±S.D.). p < 0.05; p value compared with the control group.
297 298
55.56% ± 2.56% in compare with untreated cells (42.10% ± 1.93%) and cells only treated with NAC (43.79% ± 2.32%). The results were
proposed that chimaphilin-elicited ROS triggered chimaphilininduced apoptosis of MCF-7.
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
299 300
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
(Shi, 2002). In response to proapoptotic signals, caspase cascades are activated and function, resulting in disassembly of the cell. At the same time, apoptosis are significantly regulated by the Bcl-2 protein family, which decide whether a cell will live or die. PARP, a substrate of caspase cascades, is cleaved, ultimately leading to cell death (Gross, 1999). Fig. 8A showed that the expression levels of caspase-3 and caspase-9 were decreased after treatment with different concentration of chimaphilin for 24 h. Moreover, the remarkable cleavage of caspases-3 and caspase-9 were observed in compared with control cells. The Bcl-2 protein levels decreased in MCF-7 cells, while the Bad protein levels increased. Moreover, the cleavage of PARP was also observed. Western blotting showed that the intact 116 kDa moieties of PARP were decreased and that the cleaved forms (89 kDa) were increased in MCF-7 cells treated with 40 lM chimaphilin. In contrast, MCF-7 cells were treated with 10 mM NAC for 30 mines following 40 lM chimaphilin. Western blotting showed that NAC could inhibit the decrease of Bcl-2, the increase of Bad, the cleavage of caspase-3, caspase-9 and PARP in apoptotic cells induced by chimaphilin (Fig. 8B). In a word, chimaphilin could induce apoptosis involving in ROS-mediated mitoQ2 chondrial pathway in MCF-7 cells. (see Fig. 9).
305
3.5. Chimaphilin activated caspases and the execution phase of apoptosis
4. Discussion
326
The caspase-cascade system plays crucial roles in cell apoptosis, and it is reported that caspases are the executors of apoptosis
Quinones are one of the most clinical anticancer drugs in the world, which have different active function. In apoptotic cells,
327
Fig. 5. DNA fragmentation in MCF-7 cells treated with chimaphilin. Lane 1, treatment with 0 lM chimaphilin; lane 2, treatment with 20 lM chimaphilin; lane 3, treatment with 30 lM chimaphilin; lane 4, treatment with 40 lM chimaphilin; lane 5, DNA size marker. The experiment was repeated three times.
301 302 303 304
5
Fig. 6. Mitochondrial membrane potential disruption in MCF-7 cells treated with chimaphilin. Cells were treated with different concentration of chimaphilin for 24 h. (A) Flow cytometry assay of chimaphilin-induced mitochondrial membrane potential disruption of cells. (a) treatment with 0 lM chimaphilin; (b), treatment with 20 lM chimaphilin; (c) treatment with 30 lM chimaphilin; (d) treatment with 40 lM chimaphilin. (B) Columns show mean values of three experiments (±S.D.). (C) NAC inhibited chimaphilin-induced mitochondrial membrane potential disruption of cells. (a) treatment with 0 lM chimaphilin; (b) treatment with 40 lM chimaphilin; (c) treatment with 10 mM NAC following 40 lM chimaphilin; (d) treatment with 10 mM NAC. (D) Columns show mean values of three experiments (±S.D.). p < 0.05; p value compared with the control group.
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325
328
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 6
W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
Fig. 7. ROS generation in MCF-7 cells treated with chimaphilin. Cells were treated with different concentration of chimaphilin for 24 h. (A) Flow cytometry assay of chimaphilin-induced ROS generation of cells. (a) treatment with 0 lM chimaphilin; (b) treatment with 20 lM chimaphilin; (c) treatment with 30 lM chimaphilin; (d) treatment with 40 lM chimaphilin. (B) Columns show mean values of three experiments (±S.D.). (C) NAC inhibited chimaphilin-induced ROS generation of cells. (a) treatment with 0 lM chimaphilin; (b) treatment with 40 lM chimaphilin; (c) treatment with 10 mM NAC following 40 lM chimaphilin; (d) treatment with 10 mM NAC. (D) Columns show mean values of three experiments (±S.D.). p < 0.05; p value compared with the control group.
Fig. 8. Effect of chimaphilin on apoptosis-related proteins involved in mitochondria/caspase-indepented signaling pathway in MCF-7 cells. A The expression level of caspase3, caspase-9, PARP, Bcl-2 and bad, was detected by western blotting in MCF-7 cells treated with chimaphilin for 24 h. B The cells was pretreated for 30 min by N-acety-Lcystein before treatment with chimaphilin. Then the expression levels of caspase-3, caspase-9, PARP, Bcl-2 and Bad were detected.
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
7
Fig. 9. Signaling pathway of apoptosis induced by chimaphilin in MCF-7 cells.
329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365
naphthoquinone derivatives generally lead to generation of reactive oxygen species through redox cycling, consisting with inhibiting NAD(P)H: quinone oxidoreductase. The naphthoquinone derivatives have the chemical structure of quinone with gaining electrons (Criddle et al., 2006). Menadione, a naphthoquinone, triggers apoptosis of mouse myogenic C2C12 cells through generation of reactive oxygen species, oxidative stress, iron-dependent lipid peroxidation, and glutathione depletion (Chiou et al., 2003). In U87MG glioma cells, shikonin also induces the generation of ROS, depletion of GSH, disruption of mitochondrial membrane potential, upregulation of p53, and cleavage of PARP (poly(ADP-ribose) polymerase). The changes contribute U87MG glioma cells to apoptosis (Chen et al., 2012). Chimaphilin, isolated from pyrola [Passiflora incarnata Fisch.], is also a 1,4-naphthoquinone derivative. However, there have been no reports about the antitumor mechanism of chimaphilin. In this study, we first investigated the anticancer activity of chimaphilin toward human breast cancer MCF-7 cells. In addition, we presumed that chimaphilin could trigger apoptosis of human breast cancer MCF-7 cells through the mechanism of a ROS-mediated mitochondrial pathway. In the therapies of carcinoma, apoptosis is a main approach. Apoptosis plays a crucial role in the cellular progress of proliferation, differentiation, senescence, and death. The MTT assay was carried out to evaluate the cytotoxicity of chimaphilin toward human breast cancer MCF-7 cells. Chimaphilin inhibited the growth of MCF-7 cells in a dose- not time-dependent manner (Fig. 2), but it could not inhibit the proliferation of HepG-2 cells, HCT-116 cells and SMMC-7721 cells. As known in apoptotic cells, phosphatidylserine is translocated from the inner to the outer leaflet of the plasma membrane. The stain of annexinV-FITC/PI was used to explore whether apoptosis occurred in MCF-7 cells exposed to chimaphilin. In compared with cells without treatment of chimaphilin, fluorescence intensities of cells were increased at the concentration of 30 and 40 lM chimaphilin (Fig. 4A and B). Nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation are significant characteristic of apoptosis. Chromatin condensation was observed in MCF-7 cells treated with
20 lM chimaphilin (Fig. 3). Moreover, DNA Ladder was also observed by agarose gel electrophoresis assay (Fig. 5). Consequently, it was demonstrated that chimaphilin could initiate apoptosis of human breast carcinoma MCF-7 cells. As a major compartment of energy metabolism, mitochondrial plays a pivotal role in the progress of caspase-denpendent apoptosis. When antitumor reagents stimulate cancer cells, pro-apoptotic signals regulate and accumulate the expression of pro- and antiapoptosis Bcl-2 family proteins in the cytoplasm. Then these proteins converge on mitochondria to change mitochondrial outer membrane permeabilization, which results in the release of cytochrome c, activation of caspase cascades, and apoptosis (Douglas and Kroemer, 2004; Douglas and John, 1998). In MCF-7 cells, chimaphilin caused intracellular high Bad/Bcl-2 ratio (Fig. 8A and B), which contributed to reduce mitochondrial membrane potential (Fig. 6A and B) and evoke mitochondrial outer membrane permeabilization, resulting in the release of cytochrome c from mitochondria to cytoplasm matrix. Once cytochrome c binds Apaf-1 and procaspase-9, procaspase-9 is cleaved and activates procaspase-3 (Jiang and Wang, 2004). PARP, known as a poly (ADP-ribose) synthetase and poly (ADP-ribose) transferase, catalyzes the transfer of the ADP-ribose moiety from the respiratory co-enzyme NAD+ to a limited number of acceptor proteins involved in chromatin architectures when cells are stimulated by DNA strand-breaks (Van wijk and Hageman, 2005; Huber et al., 2004). PARP is generally cleaved as a substrate by caspase-3 in apoptosis (Zhang et al., 2005). As shown in Fig. 8A and B, chimaphilin activated caspase-9, caspase-3 and cleavage of PARP in MCF-7 cells. Accordingly, we found that MCF-7 cells treated with chimaphilin were subjected to apoptosis involving in mitochondria pathway. It has been reported by many authors that ROS can induce apoptosis in many different cell systems (Simon et al., 2000). Hydrogen peroxide-induced apoptosis is required for the release of mitochondria-derived ROS and activation of caspase-3 (Dumont et al., 1999). ROS elicited by menadione via redox can induce apoptosis of murine pancreatic acinar cells (Criddle et al., 2006). Cajanol, a novel isoflavanone from pigeonpea, also triggers
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402
FCT 7930
No. of Pages 8, Model 5G
6 May 2014 8
W.-D. Ma et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
416
apoptosis through a ROS-mediated mitochondrial pathway (Meng et al., 2010). ROS from mitochondrial may oxidize membrane proteins of mitochondrial, change mitochondrial outer membrane permeabilization, and lead to disruption of mitochondrial membrane potential, which contribute to the release of cytochrome c and apoptosis (Petrosillo et al., 2003). Chimaphilin could cause ROS generation and lead to apoptosis of MCF-7 cells in a concentration-dependent manner. The intracellular ROS increase was consistent to apoptosis induced by chimaphilin in MCF-7 cells (Fig. 7A and B). NAC, a scavenging reagent of ROS, could prevent chimaphilin-induced disruption of mitochondrial membrane potential and apoptosis in MCF-7 cells (Fig. 6C, D and Fig. 4C, D). Therefore, our results suggested that the main cause of chimaphilin-induced apoptosis was ROS.
417
5. Conclusion
418
425
The present study demonstrated that chimaphilin could induce apoptosis of MCF-7 cells through a ROS-regulated mitochondrial pathway. Generation of ROS was one cause of apoptosis induced by chimaphilin. Further research is needed to pinpoint other pivotal signaling pathways induced by chimaphilin toward human breast cancer MCF-7 cells. Our results might provide the basis data for future clinical application of chimaphilin treatment in breast cancer.
426
Conflict of Interest
403 404 405 406 407 408 409 410 411 412 413 414 415
419 420 421 422 423 424
427
The authors declare that there are no conflicts of interest.
428
Transparency Document
429
The Transparency document associated with this article can be found in the online version.
430 431
432
Acknowledgements
433
438
The authors gratefully acknowledge the financial supports by, Program for Agricultural Science and Technology Achievements Transformation Fund Program (2012GB23600641), Program for Importation of International Advanced Forestry Science and Technology, National Forestry Bureau (2012-4-06), Special Fund of National Natural Science Foundation of China (31270618).
439
References
440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460
Apel, K.H., 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399. Bonilla-porras, A.R., Jimenez-del-rio, M., Velez-pardo, C., 2011. Vitamin K3 and vitamin C alone or in combination induced apoptosis in leukemia cells by a similar oxidative stress signalling mechanism. Cancer Cell Int. 11, 1–11. Chen, C.H., Lin, M.L., Ong, P.L., Yang, J.T., 2012. Novel multiple apoptotic mechanism of shikonin in human glioma cells. Ann. Surg. Oncol. 19, 3097–3106. Chiou, T.J., Chu, S.T., Tzeng, W.F., 2003. Protection of cells from menadione-induced apoptosis by inhibition of lipid peroxidation. Toxicology 191, 77–88. Criddle, D.N., Gillies, S., Baumgartner-wilson, H.K., Jaffar, M., Chinje, E.C., Passmore, S., Chvanov, M., Barrow, S., Gerasimenko, O.V., Tepikin, A.V., Sutton, R., Petersen, O.H., 2006. Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells. J. Biol. Chem. 281, 40485–40492. Crompton, M., Barksby, E., Johnson, N., Capano, M., 2002. Mitochondrial intermembrane junctional complexes and their involvement in cell death. Biochimie 84, 143–152. Douglas, R.G., John, C.R., 1998. Mitochondria and apoptosis. Science 281, 1309– 1312. Douglas, R.G., Kroemer, G., 2004. The pathophysiology of mitochondrial cell death. Science 305, 626–629.
434 435 436 437
Dumont, A., Hehner, S.P., Hofmann, T.G., et al., 1999. Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB. Oncogene 18, 747– 757. Elmore, S., 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35 (4), 495–516. Galván, I.J., Mir-rashed, N., Jessulat, M., Atanya, M., Golshani, A., Durst, T., Petit, P., Amiguet, V.T., Boekhout, T., Summerbell, R., Cruz, I., Arnason, J.T., Smith, M.L., 2008. Antifungal and antioxidant activities of the phytomedicine pipsissewa, Chimaphila umbellata. Phytochemistry 69, 738–746. Gong, K., Li, W., 2011. Shikonin, a Chinese plant-derived naphthoquinone, induces apoptosis in hepatocellular carcinoma cells through reactive oxygen species: a potential new treatment for hepatocellular carcinoma. Free Radical Biol. Med. 51, 2259–2271. Gross, A.J., 1999. BCL-2 family members and the mitochondria in apoptosis. Genes 13, 1899–1911. Hu, W.J.J., 2003. Anticancer therapy targeting the apoptotic pathway. Lancet Oncol. 4, 721–729. Huber, A., Bai, P., De murcia, J.M., et al., 2004. PARP-1, PARP-2 and ATM in the DNA damage response: functional synergy in mouse development. DNA Repair 3, 1103–1108. Jiang, X., Wang, X., 2004. Cytochrome C-mediated apoptosis. Annu. Rev. Biochem. 73, 87–106. Klyomi, K., Katsuya, T., Kiyohisa, U., Hisato, K., Tsutomu, S., Hiroshi, N., 1992. Platelet aggregation inhibitors and inotropic constituents in Pyrolae herba. Chem. Pharm. Bull. 40, 2083–2087. Lin, C.H., Huang, C.C., Wang, T.W., Wang, Y.J., Lin, P.H., 2007. Disparity in the induction of glutathione depletion, ROS formation, poly(ADP-ribose) polymerase-1 activation, and apoptosis by quinonoid derivatives of naphthalene in human cultured cells. Chem. Biol. Interact. 165, 200–210. López, E., Arce, C., Oset-gasque, M.J., Cañadas, S., González, M.P., 2006. Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture. Free Radical Biol. Med. 40, 940–951. Ly, J.D., Grubb, D.R., Lawen, A., 2003. The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis: Int. J. Programmed 8, 115– 128. Meng, L., Xia, L., Zu, Y., Fu, Y.J., Su, Z., Yao, L.P., Efferth, T., 2010. Cajanol, a novel anticancer agent from Pigeonpea [Cajanus cajan (L.) Millsp.] roots, induces apoptosis in human breast cancer cells through a ROS-mediated mitochondrial pathway. Chem. Biol. Interact. 188, 151–160. Nagata, S., 2000. Apoptotic DNA fragmentation. Exp. Cell Res. 256, 12–18. Natarajan, N., Thamaraiselvan, R., Lingaiah, H., et al., 2011. Effect of flavonone hesperidin on the apoptosis of human mammary carcinoma cell line MCF-7. Biomed. Prev. Nutr. 1, 207–215. Noori, S., Hassan, Z.M., 2012. Tehranolide inhibits proliferation of MCF-7 human breast cancer cells by inducing G0/G1 arrest and apoptosis. Free Radical Biol. Med. 52, 1987–1999. Peter, M.E., Heufelder, A.E., Hengartner, M.O., 1997. Advances in apoptosis research. Proc. Natl. Acad. Sci. 94 (24), 12736–12737, Proceedings of the National Academy of Sciences 94, 11273–12736. Petrosillo, G., Ruggiero, F.M., Paradies, G., 2003. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria. FASEB J. 17, 2202–2208. Scarlett, J.L., Sheard, P.W., Hughes, G., Ledgerwood, E.C., Ku, H.H., Murphy, M.P., 2000. Changes in mitochondrial membrane potential during staurosporineinduced apoptosis in Jurkat cells. FEBS Lett. 475, 267–272. Shi, Y., 2002. Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell 9, 459–470. Simon, H.U., Haj-yehia, A., Levi-schaffer, F., 2000. Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 5, 415–418. Sun, Y.A., 1999. Menadione-induced apoptosis and the degradation of lamin-like proteins in tobacco protoplasts. Cell. Mol. Life Sci. CMLS 55, 310–316. Sun, C., Chen, Z., Wang, W., Li, Y., Dong, Y., Wang, L., 2011. Research on the development and utilization of Pyrola resources. Northern Horticulture 1, 220– 222. Van wijk, S.J., Hageman, G.J., 2005. Poly (ADP-ribose) polymerase-1 mediated caspase-independent cell death after ischemia/reperfusion. Free Radical Biol. Med. 39, 81–90. Vinatier, D., Dufour, P., Subtil, D., 1996. Apoptosis: a programmed cell death involved in ovarian and uterine physiology. Eur. J. Obstet. Gynecol. Reprod. Biol. 67, 85–102. Wang, H., Xu, Y., Yan, J., Zhao, X., Sun, X., Zhang, Y., Guo, J., Zhu, C., 2009. Acteoside protects human neuroblastoma SH-SY5Y cells against beta-amyloid-induced cell injury. Brain Res. 1283, 139–147. Xiong, Y., Liu, X., Lee, C.P., Chua, B.H., Ho, Y.S., 2006. Attenuation of doxorubicininduced contractile and mitochondrial dysfunction in mouse heart by cellular glutathione peroxidase. Free Radical Biol. Med. 41, 46–55. Yao, X.H., Zhang, D.Y., Zu, Y.G., Fu, Y.J., Luo, M., Gu, C.B., Efferth, T., 2013. Free radical scavenging capability, antioxidant activity and chemical constituents of Pyrola incarnata Fisch. leaves. Ind. Crops Prod. 49, 247–255. Zhang, Y., Zhang, X., Park, T.S., et al., 2005. Cerebral endothelial cell apoptosis after ischemia–reperfusion: role of PARP activation and AIF translocation. J. Cerebral Blood Flow Metab. 25, 868–877.
Please cite this article in press as: Ma, W.-D., et al. Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.014
461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544
