Title:
Protein hydrolysates from tuna cooking juice inhibit cell growth and induce apoptosis of human breast cancer cell line MCF-7
Names of authors:
Chuan-Chuan Hunga,b; Yu-Hsuan Yanga; Pei-Feng Kuoa; and Kuo-Chiang Hsua,b*
Affiliation and address of authors:
a Department of Nutrition, China Medical University, No. 91, Hsueh-Shih Road,
Taichung 40402, Taiwan.
b Department of Health and Nutrition Biotechnology, Asia University, No. 500,
Lioufeng Road., Taichung 41354, Taiwan. Short title:
Peptides induce apoptosis of MCF-7 *Corresponding author
Tel.: +886 4 22053366 ext 7522; fax: +886 4 22062891 E-mail address：[email protected] (K. C. Hsu) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Abstract
The effects of peptides from tuna cooking juice hydrolysates by Protease XXIII (PA) on cell growth and induction of apoptosis of human breast cancer cell line MCF-7 were determined. The PA hydrolysates showed antiproliferative activities up to 25% against MCF-7 cells, and the >2.5 kDa ultrafiltration fraction (PAH2.5) possessed the highest antiproliferative activity with an IC50 value of 1.39 mg/mL. PAH2.5 induced cell cycle arrest in S phase through the increases of p21 and p27, and decrease of cyclin A expression. Further, PAH2.5 also induced apoptosis of MCF-7 cells by downregulation of the expression of Bcl-2, PARP and caspase 9, and upregulation of the expression of p53, Bax and cleaved capase 3. Two peptides were identified in PAH2.5 as KPEGMDPPLSEPEDRRDGAAGPK (2449.292 Da) and KLPPLLLAKLLMSGKLLAEPCTGR (2562.405 Da). Thus, tuna cooking juice may be a good protein source of antiproliferative peptides against MCF-7.
Keywords: tuna cooking juice; peptides; breast cancer; antiproliferation; cell cycle; apoptosis 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
1. Introduction
Breast cancer is now the most common cause of female cancer and leading cause of cancer deaths among women in the United States and many other parts of the world (Ferlay, Shin, Bray, Forman, Mathers, & Parkin, 2010; Jemal, Siegel, Xu, & Ward, 2010). In Taiwan, breast cancer is the most leading incidence and the 4th cause of death from female cancer, and, in recent years, its mortality rate has increased and average age of death decreased.
Tuna is one of the most important fisheries in Taiwan, and its production and output value per year are over 300,000 tonnes and 31 billion NT dollars (approximately US$ 1 billion), respectively. Tuna cooking juice, a byproduct during the processing of canned tuna, contains approximately 5% proteins containing about 30% hydrophobic amino acids (Jao, & Ko, 2002; Huang, Jao, Ho, & Hsu, 2012) and is always discarded. Our research group has determined that tuna cooking juice possessed some physiological functions, such as antioxidative (Hsu, Lu, & Jao, 2009), antihypertensive (Hsu, Cheng, & Hwang, 2007) and dipeptidyl peptidase IV (DDP-IV) inhibitory (Huang et al., 2012) activities.
Peptides derived from various protein sources were also investigated to show antitumour or antiproliferative activities against cancer cells. Two peptides (dermaseptins B2 and B3) in the skin secretions of the South American tree frog 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
inhibited the proliferation of the human prostatic adenocarcinoma PC-3 cell line with an EC50 of 2-3 μM (van Zoggel et al., 2012). FF/CAP18, an analogue peptide derived from an endogenous cathelicidin family member, showed antiproliferative activity against colon cancer cell line HCT116 with the loss of mitochondrial membrane potential, and resulted in the early stage of apoptosis (Kuroda et al., 2012). Lunasin from soybeans, was found to suppress chemical carcinogen and viral oncogene-induced transformation of mammalian cells and inhibit skin carcinogens in mice (Galvez, Chen, Macasieb, & de Lumen, 2001; Jeong, Jeong, Kim, & de Lumen, 2007). A hydrophobic peptide, X-MLPSYSPY (1,157 Da) from defatted soy protein hydrolyzed with thermoase showed in vitro cytotoxicity on mouse monocyte macrophage cell line (Kim, Kim, Kim, Kang, Woo, & Lee, 2000). In our previous study, two hydrophobic peptides, LPHVLTPEAGAT (1,206 Da) and PTAEGGVYMVT (1,124 Da) isolated from tuna dark muscle byproduct had a dose-dependent inhibition effect on human breast cancer cell line MCF-7 (Hsu, Li-Chan, & Jao, 2011). A study has revealed that modulation of hydrophobicity of peptides plays a crucial role against cancer cells (Huang, Wang, Wang, Liu, & Chen, 2011). Therefore, protein source with high contents of hydrophobic amino acids may have the potential to possess anticancer and antiproliferative activities against cancer cells. On the other hand, peptides were reported to be able to induce apoptosis in tumour 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71
cells and as prime candidates for the development of anticancer therapeutics (Bhutia & Maiti, 2008).
It is surprising that there were only few related research reports on antiproliferative and apoptosis of cancer cell lines induced by peptides obtained from food proteins, especially fish proteins. In this study, we tried to use commercial proteases, Protease XXIII (PA) to hydrolyze tuna cooking juice and then identify the antiproliferative activity on the human breast cancer cell line MCF-7. In addition, we investigated the effects of the hydrolysates on cell cycle and apoptosis of MCF-7, and the amino acid sequences of the peptides in the hydrolysates were also identified.
2. Materials and methods 2.1. Sample preparation
A canned tuna processor in Chiayi County (Taiwan) supplied the tuna cooking juice in which the protein content was 4.71% (data not shown). The whole tuna fish (Thunnus tonggol) was cooked by steam (100-105℃) for 3-4 h, after which, the hot collected cooking juice was sealed in 400 mL polyethylene bags and then transferred to our laboratory immediately, and stored at 4℃ overnight. The cooking juice was filtrated through two layers of gauze to remove floating fats and solids, and the filtrate was collected and stored at -20℃ until used within 3 months.
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2.2. Chemicals and reagents
Protease XXIII (PA) (specific activity of 3.8 units/mg solid), an endopeptidase prepared from Aspergillus melleus, was obtained in dry powder form from Sigma-Aldrich, Inc. (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-diphenyl tetrazolium bromide (MTT), L-glutamine, L-leucine and 2,4,6-trinitrobenzenesulphonic acid (TNBS) were purchased from Sigma-Aldrich, Inc. Other chemicals and reagents used were analytical grade and commercially available.
2.3. Enzymatic hydrolysis
Twenty-five millilitres of tuna cooking juice were adjusted to pH 7.5 by using 2 M NaOH and then incubated at 37℃ in a water bath for 20 min prior to enzymatic hydrolysis. The hydrolysis reaction was started by the addition of enzyme solutions at the enzyme/substrate (E/S) ratio of 2.1% (25 mg enzyme powder dissolved in 1 mL ddH2O). After hydrolysis up to 6 h, the hydrolysate solutions were heated in a boiling water for 15 min to inactivate enzymes and then cooled in cold water at room temperature for 20 min. Hydrolysates were adjusted to pH 7.0 with 2 M NaOH and centrifuged (Centrifuge 05P-21, Hitachi Ltd., Katsuda, Japan) at 10,000g and 4℃ for 10 min. The supernatant was lyophilised and stored at -20℃.
2.4. Degree of hydrolysis 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111
The degree of hydrolysis (DH) of the hydrolysate was determined as the ratio of the amount of amino acid released during hydrolysis to the maximum amount of α-amino acid in tuna cooking juice (Benjakul & Morrissey, 1997). Properly diluted samples (125 μL) were mixed with 2 mL of 0.2125 M sodium phosphate buffer (pH 8.2), followed by the addition of 1 mL of 0.01% TNBS. The mixtures were then incubated in a water bath at 50℃ for 30 min in the dark. The reaction was terminated by the addition of 2 mL of 0.1 M sodium sulphite. The mixtures were cooled down at ambient temperature for 20 min. The maximum amount of α-amino acid in tuna cooking juice was obtained by acid hydrolysis with 6 M HCl at 105℃ for 24 h. The acid-hydrolysed sample was then filtered through Whatman filter paper No. 1 to remove the unhydrolysed debris. The supernatant was neutralised with 6 M NaOH before amino acid determination. The absorbance was measured at 420 nm and α-amino acid was expressed in terms of L-leucine. The DH was calculated as follows: DH (%) = [(Lt-Lo)/(Lmax-Lo)] x 100, where Lt is the amount of α-amino acid released at time t; Lo is the amount of α-amino acid in original tuna cooking juice; Lmax is the maximum amount of α-amino acid in tuna cooking juice (Beak & Cadwallader, 1995). 2.5. Cell culture 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130
Human breast cancer MCF-7 cells and MCF-10A mammary epithelial cells purchased from Bioresource Collection and Research Center (BCRC) (Hsinchu, Taiwan) were cultured in a 37℃ humidified atmosphere with 5% CO2 in DMEM, supplemented with 10% FBS, 1% PSN and 1.5 g/L sodium bicarbonate (pH 7.1-7.2).
2.6. MTT assay
To avoid pH variation of the cell culture medium during sample solubilisation, fish hydrolysate stock solution was prepared in 0.1 M PBS (pH 7.4). The cells were seeded in a 96-well microtiter plate (1 x 104 cells/well) overnight, and then treated with various concentrations of hydrolysates and their ultrafiltration fractions. After incubating for 72 h, the effect of hydrolysates on cell growth was examined by the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay. About 20 μL of MTT solution (5 mg/mL, Sigma Chemical Co.) were added to each well and incubated at 37℃ for 4 h. The supernatant was aspirated and the MTT-formazan crystals formed by metabolically viable cells were dissolved in 200 μL of isopropanol. Finally, the absorbance was read at 570 nm with a microplate reader. The hydrolysate concentration which gives 50 % growth inhibition is referred to as the IC50. 2.7. Ultrafiltration (UF) 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150
The peptides of the hydrolysates were fractionated by ultrafiltration (model ABL085, Lian Sheng Tech. Co., Taichung, Taiwan) with spiral wound membranes having molecular mass cutoffs of 2.5 and 1 kDa. The fractions were collected as follows: >2.5 kDa, peptides retained without passing through 2.5 kDa membrane; 1-2.5 kDa, peptides permeating through the 1-2.5 kDa membrane but not the 1 kDa membrane; <1 kDa, peptides permeating through the 1 kDa membrane. All fractions collected were lyophilized and stored in a desiccator until use.
2.8. Cell Cycle
For cell cycle analysis, cells were seeded at a density of 1×105 cells/well in 6-well plates, cultured overnight, and then treated with various concentrations of PA hydrolysates. To analyze the cell cycle, after 72 h of treatment the cells were harvested by trypsinisation, washed in PBS, and fixed in 70% ice cold ethanol. The cell pellets were resuspended in 500 μL of a solution containing 50 μg/mL propidium iodide, 0.4 mg/mL RNase A, 0.1% Triton-X-100 in PBS buffer, and then incubated at 37℃ for 30 min. The stained cells were subjected to DNA content/cell cycle analysis using an LSR flow cytometer.
2.9. Apoptosis
For apoptosis, the FITC Annexin V Apoptosis Detection Kit (BD PharmingenTM, 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170
San Jose, CA, USA) was used to assess annexin V-positive cells. Wash cells twice with cold PBS and then resuspend cells in 1X annexin binding buffer. One hundred microliters of the solution was transferred to a 5 mL culture tube and add 5 μL of FITC Annexin V and 5 μL PI. Gently vortex the cells and incubate for 15 min at room temperature in the dark. After incubation, 400 μL of 1X annexin binding buffer were added to each tube and the cells were analyzed by flow cytometry using an LSR flow cytometer (BD Biosciences Inc., Franklin Lakes, NJ, USA).
2.10 Identification of amino acid sequence by MALDI-TOF/TOF MS/MS
The purified peptides were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), using a delayed extraction source and a 335 nm pulsed nitrogen laser. This analysis was carried out using a MALDI-TOF/TOF (UltraFlexIII, Bruker Daltonics Inc., Billerica, MA, USA). Peptides solution (0.6 μL) was mixed with 0.6 μL of saturated α-cyano-4-hydroxycinnamic acid, and a droplet of the resulting solution was placed on the sample target mass spectrometer. The droplet was dried by evaporation at room temperature and then loaded into the mass spectrometer for analysis. The instrument was operated in positive ion reflection mode with the source voltage set at 20 kV. All spectra were the results of signal averaging of 200 shots. Measurements were 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189
determined in the mass range m/z 200-4000 Da, while the peptide sequencing was determined by MS/MS spectra processing, using BioTools (Version 3.2; Bruker Daltonics Inc., Billerica, MA, USA).
2.11 Statistical analysis
Each data point represents the mean of three samples was subjected to analysis of variance (ANOVA) followed by Duncan’s multiple range test, and the significance level of P＜0.05 was employed.
3. Results and discussion 3.1. Degree of hydrolysis
The DH of tuna cooking juice hydrolyzed with PA increased dramatically during the initial 1 h and 2 h, and increased gradually thereafter (Fig. 1A). The highest DH (%) of PA hydrolysates was 14.0% after the 6-h hydrolysis. The trend and curve shape of the hydrolysis are similar to those reported in our previous studies (Hsu et al., 2009, 2011; Huang et al., 2012) and also to other studies on enzymatic hydrolysis of various protein sources (Bougatef et al., 2010; Dong, Zeng, Wang, Liu, Zhao, & Yang, 2008; Klompong, Benjakul, Kantachote, & Shahidi, 2007).
3.2. Antiproliferative activities of hydrolyates 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209
In our preliminary test, PA hydrolysates showed greater antiproliferative activity than the other two hydrolysates obtained from the hydrolysis by orientase 90N (Hankyu Bioindustry Co., Osaka, Japan) and alcalase (Novozymes North America Inc., Salem, NC, USA). In the present study, therefore, only PA hydrolysates were used for the further determinations.
The antiproliferative effect of the hydrolysates (concentration of 1 mg/mL) derived from tuna cooking juice on breast cancer cell, MCF-7, after incubation for 72 h was investigated. As depicted in Fig. 1B, all the PA hydrolysates during 1-6 h hydrolysis possessed significant antiproliferative activity as compared to the control (p<0.05). Stronger antiproliferative activities (22-25%) were observed in PA hydrolysates for 1, 2 and 4 h, but there were no significant differences between those of the 3 hydrolysates (p>0.05). No correlation exhibited between degree of hydrolysis and antiproliferative activity in this study, and this might imply the antiproliferative peptides were independent of molecular weight (Picot et al., 2006). For further purification and investigation, PA hydrolysate for 1-h hydrolysis (PAH) was chosen based on time saving principle.
3.3. Antiproliferative activity of UF fractions of hydrolysates
The antiproliferative activities of the UF fractions (>2.5 kDa, 1-2.5 kDa, and <1 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228
kDa) of PAH are shown in Fig. 2. The peptides within the >2.5 kDa UF fraction (PAH2.5) had the greatest antiproliferative activity of 37.8% (P<0.05), whereas those within the <1 kDa and 1-2.5 kDa UF fractions (PAH1 and PAH1-2.5) displayed the inhibition rates of only 20.0 and 22.8%, respectively (Fig. 2A). To the best of our knowledge, the antiproliferative peptides derived from fish protein sources were reported to have molecular weight (MW) of 440.9 Da from anchovy sauce (Lee, Kim, Lee, Kim, & Lee, 2003; Lee, Lee, Kim, Kim, & Lee, 2004) and those of 1,206 and 1,124 Da from tuna dark muscle (Hsu et al., 2011). The MWs of the antiproliferative peptides obtained from soy protein and algae protein wastes were 1,157 and 1,309 Da, respectively (Kim et al., 2000; Sheih, Fang, Wu, & Lin, 2010). However, some studies reported the MWs of the antiproliferative peptides derived from various sources to be greater than 1,400 Da. An antifungal peptide with MW of approximately 3.9 kDa, isolated from buckwheat seeds, possessed antiproliferative activities against leukaemia (L1210), breast (MCF-7), liver embryonic (WRL68) and liver (HepG2) cancer cells (Leung & Ng, 2007); and lunasin, a cancer-preventive peptide with MW of about 5.45 kDa, isolated from soy and barley, has been demonstrated to be effective against chemical carcinogens and oncogenes in mammalian cells and in a skin cancer mouse model (de Lumen, 2005). These findings demonstrate that there is no correlation between antiproliferative activity and MW of peptides.
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Fig. 2B shows the antiproliferative activity of PAH2.5 at various concentrations (0.5-5 mg/mL). The inhibition rates ranged from 30.9 to 81.4% in a dose-dependent manner; and the IC50 value against MCF-7 of PAH2.5 was 1.39 mg/mL. The peptides within the PAH2.5 at the concentrations between 1 and 10 mg/mL did not show any cytotoxic effect on the cell viability of MCF-10A mammary epithelial cells (Fig. S1). This result is similar to the study reported that the bitter melon extract inhibited breast cancer cell line MCF-7 and did not show any cytotoxic effect on human primary mammary epithelial cells, HMEC (Ray, Raychoudhuri, Steele, & Nerurkar, 2010). The IC50 value of PAH2.5 in this study was lower than Huaier aqueous extract (IC50 = 4 mg/mL) (Zhang, Kong, Yan, Yuan, & Yang, 2010), therefore, the results indicated that the PA hydrolysates would be a good source for the preparation antiproliferative peptides.
3.4. Cell cycle
As the regulation of cell cycle is critical for the growth and development of cancer, we determined the effect of PAH2.5 on cell cycle progression. The result shown in Fig. 3A indicates that the treatment of PAH2.5 (concentrations of 0, 0.5, 1 and 1.5 mg/mL) with MCF-7 for a total of 72 h caused a concentration-dependent accumulation of cells in the S phase, and a corresponding decrease in G0/G1 and 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266
G2/M-phase fractions. As summarized in Fig. 3B, the cells in the S phase increased by 21.79, 37.20, 41.38 and 47.96 % at concentrations of 0, 0.5, 1 and 1.5 mg/mL of PAH2.5, respectively. Moreover, this correlated with the decreased population in G0/G1, 71.87, 59.43, 54.75 and 50.22 %, and in G2/M, 6.33, 4.41, 3.27 and 5.44 %, respectively. These results revealed that the PAH2.5 induced cell cycle of MCF-7 arrest in S phase.
To evaluate the role of cell cycle-regulating proteins in the MCF-7 treated with PAH2.5, proteins were extracted from the PAH2.5-treated cells at 72 h for western blot analysis. When compared with the control group, the expression of cyclin A was significantly decreased in a concentration-dependent manner for the PAH2.5 treatment; whereas the expressions of p21 and p27 were increased at 0.5 and 1 mg/mL, but decreased at 1.5 mg/mL (Fig. 4). These results suggest that PAH2.5 induces cell cycle arrest in S phase through increases of p21 and p27 proteins expression and the decrease of cyclin A expression. This phenomenon is similar to those in other studies, reporting that retigeric acid B, quinacrine and evodiamine induced cancer cell cycle arrest in S phase (Liu, Liu, Xu, Young, Yuan, & Lou, 2010; Preet et al., 2012; Zhang, Fan, Xu, Yang, Wang, & Liang, 2010).
3.5. Apoptosis 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285
Since cell apoptosis may be one of the consequences of cell-cycle arrest, we examined whether PAH2.5 induced apoptosis in MCF-7 cells. We stained the cells with FITC Annexin V and PI, and we conducted internucleosomal DNA fragmentation assays. Fig. 5A shows that after the treatment of PAH2.5 for 72 h with various concentrations (0, 0.5, 1 and 1.5 mg/mL), the percentages of apoptotic cells were 220, 277 and 349 % for MCF-7 cell lines as compared to the control with the baseline of 100% (Fig. 5B). These results indicated that PAH2.5 induced apoptosis of MCF-7 cell lines in a concentration-dependent manner.
The expression of apoptosis-related proteins was investigated in order to analyze the underlying mechanisms by Western blot analysis. As shown in Fig. 6, PAH2.5 downregulated the expression of Bcl-2, PARP and caspase 9, and upregulated the expression of p53, Bax and cleaved caspase 3 at the concentrations of 0.5 and 1 mg/mL. These results suggested that PAH2.5 induced apoptosis of MCF-7 cells by activating caspase-related proteins family and might be through mitochondria mediated pathway. This is in agreement with a previous study of activating p53 (Sax, Fei, Murphy, Bernhard, Korsmeyer, & El-Deiry, 2002; Vogelstein, Lane, & Levine, 2000; Yamaguchi, Chen, Bhalla, & Wang, 2004) and triggering relocalisation of cytochrome c lead to apoptosis of cancer cells (Pan, Becker, & Gerhauser, 2005; Szigeti et al., 2010). 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304
3.6. Amino acid sequences of peptides in PAH2.5
PAH2.5 was used to identify the amino acid sequences of peptides by MALDI TOF/TOF MS/MS. Two peptides with the molecular mass of 2449.292 and 2562.405 were determined. After the analysis by MS/MS spectra processing with BioTools database, the amino acid sequences of the two peptides are KPEGMDPPLSEPEDRRDGAAGPK and KLPPLLLAKLLMSGKLLAEPCTGR, respectively (Fig. S2). The both peptides are rich in hydrophobic amino acids, such as Pro, Leu, Ala and Phe. A previous study revealed that peptide with greater hydrophobicity showed strong anticancer activity against cancer cell lines, including MCF-7 (Huang et al., 2011). Therefore, PAH2.5 showed great antiproliferative activity of MCF-7 probably attributed to the high hydrophobicity of the peptides.
4. Conclusions
PAH2.5 showed antiproliferative effect, induced cell cycle arrest in S phase and apoptosis against MCF-7 cells. Two peptides in PAH2.5 with the molecular mass of 2449.292 and 2562.405 and their amino acid sequences were also identified. This study has clearly demonstrated that tuna cooking juice has the potential to be a valid protein source of bioactive peptides with the antiproliferative effect on MCF-7 cells and without affecting normal breast epithelial cells.
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Acknowledgments
This study was financially supported by National Science Council, Taiwan, Republic of China, with the grant No. NSC 99-2313-B-039-001-MY3.
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Figure Legends
Fig. 1. (A) Degree of hydrolysis (DH) of tuna cooking juice during hydrolysis. (B) Effect of protein hydrolysates derived from tuna cooking juice on cell proliferation of MCF-7 cells cultured for 72 h in cell culture medium containing 1 mg/mL of hydrolysates. Bars represent standard deviations from triplicate determinations. Different letters indicate the significant differences (P < 0.05)
Fig. 2. (A) Cell proliferation of MCF-7 treated with the UF fractions of PAH for 72 h at the concentration of 1 mg/mL. (B) Cell proliferation of MCF-7 treated with PAH2.5 for 72 h at various concentrations. Bars represent standard deviations from triplicate determinations.
Fig. 3. Effect of PAH2.5 on the cell cycle progression of MCF-7. (A) After the treatment of PAH2.5 at various concentrations for 72 h, MCF-7 cells were harvested, stained with propidium iodide, and analysed by flow cytometry. Flow cytometric histograms are representative of three separate experiments. (B) Quantification of percentage of MCF-7 cells treated by PAH2.5 in cell cycle. Bars represent standard deviations from triplicate determinations. Different letters indicate the significant differences (P < 0.05) 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468
Fig. 4. Effect of PAH2.5 on cell cycle-related protein levels in MCF-7 cells investigated by Western blot analysis. Cells were treated with 0.5, 1 and 1.5 mg/mL of PAH2.5 for 72 h. β-actin was used as a loading control. Figures showed the representative blots from one of three experiments that gave similar results.
Fig. 5. Apoptosis assessment of MCF-7 cells treated with indicated concentrations of PAH2.5. (A) Flow cytometric analysis of PS externalization (annexin V binding) and cell membrane integrity (PI staining). Cells were treated with 0, 0.5, 1 and 1.5 mg/mL of PAH2.5 for 72 h. The dual parameter dot plots combining annexin V-FITC and PI fluorescence show the vial cell population in the lower left quadrant (Q3), the early apoptotic cells in the lower right quadrant (Q4), and the late apoptotic cells in the upper right quadrant (Q2). (B) Percentages of apoptotic cells (Q2 + Q4). Bars represent standard deviations from triplicate determinations. Different letters indicate the significant differences (P < 0.05).
Fig. 6. Effect of PAH2.5 on apoptosis-related protein levels in MCF-7 cells investigated by Western blot analysis. Cells were treated with 0.5, 1 and 1.5 mg/mL PAH2.5 for 72 h. β-actin was used as a loading control. Figures show the representative blots from one of three experiments that gave similar results.
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Fig. S1. Cell proliferation of MCF-10A treated with PAH2.5 for 72 h at various concentrations. Bars represent standard deviations from triplicate determinations.
Fig. S2. MALDI-TOF/TOF MS/MS spectrum of the peptides in PAH2.5. 489 490 491 492 493 494
