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Effect of rapeseed peptide on DNA damage and apoptosis in Hela cells
Experimental and Toxicologic Pathology 62 (2010) 519–523
Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Effect of rapeseed peptide on DNA damage and apoptosis in Hela cells Zhaohui Xue a,b,1, Zhiwei Liu c,1, Moucheng Wu b, Shiwen Zhuang a, Wancong Yu d, a
School of Agriculture and Bioengineering, Tianjin University, Tianjin 300072, PR China Department of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China National Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, PR China d Department of Bioscience and Technology, Tsinghua University, Beijing 100084, PR China b c
a r t i c l e in fo
abstract
Article history: Received 16 December 2008 Accepted 30 June 2009
Rapeseed peptide (RSP), obtained by hydrolyzing rapeseed protein, has anticancer activity. In this study, the effects of RSP on proliferation rate, morphological changes, DNA damage, cell cycle distribution and apoptosis in human cervical carcinoma (Hela) cells were investigated. RSP treatment at a concentration of 640 mg/L for 4 days inhibited Hela cell proliferation significantly, as determined by the MTT assay. We observed a dose-dependent increase in cytotoxicity induced by RSP at 20–640 mg/L. After 4 days of 320 mg/L RSP treatment, typical apoptotic changes were observed by transmission electron microscopy (TEM). Using the comet assay, we found dramatic comet tails, indicating DNA damage by RSP (20–640 mg/L). Moreover, RSP treatment caused inhibition of Hela cell growth, with cycle arrest in the S phase and apoptosis induction. Taken together, the results suggested that rapeseed peptide could be a potential antitumor compound with an apoptotic mode of action. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Apoptosis Rapeseed peptide (RSP) Hela cells Cytotoxicity Comet assay Cell cycle
Introduction Rapeseed peptide (RSP) belongs to a group of polypeptides that are obtained following hydrolysis of the rapeseed protein and have anticancer, immunomodulatory, antioxidant, antihypertensive and HIV protease inhibitory effects (Zeng et al., 2002; Xue et al., 2005, 2006, 2007a, 2007b; Maria et al., 2004; Marczak et al., 2003). In vitro and in vivo studies have demonstrated that RSP is an effective anticancer compound. In vivo, RSP can dosedependently decrease tumor weight and increase the amount of splenocytes, proliferation of activated T cells, NK activity and TNF levels in S180-bearing mice (Zeng et al., 2002; Xue et al., 2007b). In vitro, 20 mg/L RSP inhibited the growth of S180 cells and decreased membrane fluidity (Zeng et al., 2004). Apoptosis is the process of programmed cell death, and is tightly controlled. It plays important roles in many normal processes, ranging from fetal development to adult tissue homeostasis (Reed, 2001). During apoptosis, the nucleus and cytoplasm condense to produce membrane-bound apoptotic bodies that are phagocytosed by macrophages or adjacent cells. Emerging evidence has demonstrated that the anticancer activities of certain chemotherapeutic agents involve the induction of apoptosis, which is regarded as the most effective method of managing cancer (Hengartner, 2000; Hsu et al., 2004).
Cervical carcinoma is one of the most common neoplastic diseases affecting women, second only to breast cancer, with almost half a million new cases diagnosed annually, worldwide (Franco et al., 2003). Therefore, the development of chemotherapeutic/chemopreventive agents against cervical carcinoma is vital to reducing the prevalence and mortality of this disease. The main aim of this paper was to study the cytotoxicity of RSP. To do this, we investigated the effects of RSP treatment on DNA damage, the stage of cell cycle arrest, and apoptosis in human cervical carcinoma (Hela) cells.
Materials and methods RPMI-1640, fetal calf serum (FCS), penicillin G, streptomycin and trypsinase were obtained from Invitrogen (Carlsbad, CA). Dimethyl sulphoxide (DMSO), propidium iodide (PI), ethidium bromide (EB), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), Fluorouracil (5-FU) (Jinyao company, Tianjin, China), phosphate-buffered saline (PBS, pH 7.2–7.6) and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO). Normal melting point agarose (NMA) and low melting point agarose (LMA) were purchased from the Huamei Company (Beijing, China). Cell culture
Corresponding author. Tel.: +86 10 62773664.
E-mail address: [email protected] (W. Yu). These authors contributed equally to this work.
1
0940-2993/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.06.013
Hela cells were supplied by the Chinese Center for Disease Control and Prevention (China CDC). They were maintained as a
520
Z. Xue et al. / Experimental and Toxicologic Pathology 62 (2010) 519–523
monolayer culture at 37 1C and 5% CO2 in RPMI-1640 supplemented with 10% FCS, 10 U/mL penicillin, 10 mg/mL streptomycin, 200 mg/L L-Glutamine (Sigma) and 5 ml/L insulin (Sigma). Preparation of RSP RSP (rapeseed peptide), hydrolyzed from rapeseed protein, was isolated with alcalase and flavourzyme in our laboratory. RSP was prepared as a stock solution of 3200 mg/L in basal medium RPMI1640 and kept at 20 1C. The stock solution was diluted to appropriate final concentrations with the culture medium prior to use in all experiments. Control cultures received the same volume of RPMI-1640. MTT cytotoxicity assay The cytotoxicity induced by RSP was measured by the MTT assay. Briefly, Hela cells were plated in 96-well culture plates (5.0 104 cells per well). After a 24 h incubation period, the cells were treated with RSP (20–640 mg/L) for 2, 4, and 6 days. 160 mg/L Fluorouracil (5-FU) was selected as positive control. The medium was replaced every other day. Dye solution (10 mL), specific for the MTT assay, was added to each well for an additional 4 h at 37 1C. After adding the DMSO stop solution (100 mL/well), the absorbance (A) at 620 nm was measured by an ELISA reader (Bio-TEK, USA). The percent viability of the treated Hela cells was calculated as follows: (A620 nm) sample/(A620 nm) control 100%. Morphological observation by TEM Hela cells were cultured with basal medium and medium containing 320 mg/L RSP for 4 days. Then, the cells were examined by TEM (transmission electron microscope; Philips EM 208 s) in the following procedure: the monolayer cells were detached by trypsinization and washed twice with PBS. The cell pellets were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde at 4 1C for 2 h. The cells were then washed three times with cacodylic acid sodium salt buffer (pH 7.2) and postfixed in 1% osmic acid at 4 1C for an additional 2 h. After dehydration with gradient ethanol and 1,2-epoxypropane (twice for 15 min each). The samples were first placed in 1,2-epoxypropane and resin (V/V, 1:1) for 1 h, followed by 1,2-epoxypropane and resin (V/V, 1:4) for another hour, and then in 100% resin at room temperature for 2 h. Finally, Epon812 resin was solidified at 35 1C for 16 h, 45 1C for 8 h, 55 1C for 14 h and 60 1C for 48 h. Ultrathin sections were stained with uranyl acetate and lead citrate for examination by TEM. The control groups, normal and anoxic Hela cells, were similarly prepared. Comet assay The comet assay, also called the single cell gel electrophoresis (SCGE), is a simple, rapid and sensitive method for measuring DNA strand breaks at the level of individual eukaryotic cells (Collins, 2004). The comet assay was performed following the alkaline procedure described by Singh et al. (1988), with minor modifications. Slides were cleaned with 100% ethanol and air-dried. Two solutions containing 0.5% normal melting agarose (NMA) and 0.5% low melting agarose (LMA) were made. Briefly, 120 mL of 0.5% NMA at 45 1C was transferred onto a pre-cleaned slide, spread evenly, and placed at room temperature, allowing the agarose to solidify. Afterwards, 75 mL of 0.5% LMA at 37 1C with 5 mL of cell suspension (3 106 cells/mL) were applied and covered with a coverslip. Subsequently, slides were kept at 4 1C for up to 5 min, to allow the agarose to solidify. The coverslip was then carefully
removed. Finally, a third layer, consisting of 80 mL of 0.5% at 37 1C, LMA was added. After solidification, slides were immersed in icecold, freshly prepared lysis solution (1% sodium sarcocinate, 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10.0, 1% Triton X-100, 10% DMSO) at 4 1C for at least an hour. Then, slides were placed in a horizontal electrophoresis device filled with freshly prepared electrophoresis buffer (1 mM Na2EDTA, 300 mM NaOH) and subjected to 25 V and 300 mA for 15 min. Afterwards, slides were neutralized with 0.4 M Tris–HCl (pH 7.5), stained with 20 mL of ethidium bromide (EB, 10 mg/mL) and kept in a moist chamber in the dark at 4 1C until analysis. All the above steps were carried out under a red light to prevent any additional DNA damage. EB-stained nucleotides were viewed with a fluorescence microscope at 200 magnification using blue light excitation. The comet’s tail length was measured from the trailing edge of the nucleus to the leading edge of the tail using a calibrated slide in the ocular of the microscope. The absolute length in micrometers was calculated using a calibrated stage micrometer. A minimum of 50 cells were counted for each sample. Analysis of cell-cycle phase distribution by flow cytometry PI is a highly water soluble, fluorescent compound that cannot pass through intact membranes and is generally excluded from viable cells. It binds to DNA by intercalating between the bases in double stranded nucleic acids of exposed nuclei (Lima et al., 2002). After fixed and permeabilized cells were stained with PI, the relative fluorescence intensity of PI was measured by flow cytometry as an indirect measure of the cellular DNA content. After 4 days of culture with medium containing 20, 80, 320, 640 mg/L RSP, Hela cells were collected by trypsinization, washed with PBS and fixed in ice-cold 70% ethanol for at least 24 h. The cells were washed twice with PBS by centrifuging at 1000 rpm for 10 min. The cells were resuspended in 3 ml of PBS for 5 min. Then, the cells were stained with 1 ml PI (0.1 mg/mL with 0.1% TritonX100) and incubated in the dark for 30 min. The samples were analyzed by flow cytometry (FACSCalibur, BD, USA). Cells with a lower DNA content than that of the G1 phase of the cell cycle were considered as hypodiploid cells (sub-G1 phase). The percentage of cells in a particular phase was analyzed using the CellQuest and Modfit software (Beckman, USA). A flow cytometer equipped with a 15 mW argon laser and an excitation line of 488 nm was used. Fluorescence intensity of PI was detected at an emission wavelength 630 nm. Statistical analysis The results are expressed as mean7standard deviation (SD). The difference between control and RSP-treated cells was evaluated using the Student’s t-test. P values less than 0.05 were considered statistically significant.
Results MTT cytotoxicity assay Hela cells were cultured in 10% FCS-containing medium with or without RSP for 6 days. 20–640 mg/L RSP were selected according to the results of pre-experiments. Cytotoxicity was then evaluated using the MTT test. Under our experimental conditions, a dramatic decrease in proliferation was observed 4 days after RSP treatment (20, 80, 320 and 640 mg/L) (Fig. 1). The greatest inhibition was seen after 4 days of treatment with 640 mg/L (40%, Po0.05).
Z. Xue et al. / Experimental and Toxicologic Pathology 62 (2010) 519–523
Morphological observation by TEM As observed by TEM, the karyon was bigger and smoother in normal Hela cells (Fig. 2) than in cells treated with 320 mg/L RSP (Fig. 3). Many ‘‘apoptotic bodies’’ were observed in the RSP-treated cells. The compact masses of chromatin aggregated along the nuclear membrane. Round, compact granular masses appeared near the center of the nucleus and there was a reduction in nuclear volume. At the same time, the cytoplasm displayed condensation. Some of the nuclei degenerated into discrete spherical or ovoid fragments of highly condensed chromatin. Compared with normal (Fig. 2) and anoxic Hela cells (Fig. 4), RSP-treated cells exhibited dramatic morphological changes, characteristic of apoptotic cells. These results demonstrated that RSP induced apoptosis in Hela cells.
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damage, was measured using an ocular micrometer. The results obtained are summarized in Table 1. Typically, normal Hela cells showed comets with bright heads and small tails, while 640 mg/L RSP-treated cells exhibited comets with small heads and long diffuse tails, showing the increase of the comet tail length from 0.51 to 47.18 mm. Comets with intermediate characteristics were observed in Hela cells treated with 80, 320 and 640 mg/L RSP. A gradual increase in the tail length of comets occurred with increasing concentrations of RSP, indicating increasing DNA damage.
Analysis of cell-cycle phase distribution by flow cytometry Cell-cycle phase distribution was analyzed by flow cytometry with PI staining (Fig. 5). The percentages of cells in G0/G1, S and
Comet assay Undamaged cells had intact nuclei without tails, unlike damaged cells. The length of the comet’s tail, representing DNA
120% 0mg/L 20mg/L 80mg/L
Proliferation
100%
320mg/L
80%
640mg/L
60%
40% 0
2
4
6
8
Time (days) Fig. 1. Effect of RSP on Hela cell growth. After 24 h of adherence, Hela cells were cultured in 10% FCS medium and treated with RSP (20–640 mg/L) for 2–6 days. Results are presented as the proliferation ratio of RSP-treated Hela cells. Values were expressed as means7SD of six experiments (n ¼ 6) (P-value relative to control group: Po0.05).
Fig. 3. Morphological observation of Hela cells incubated in medium containing 320 mg/L RSP (8300 ).
Fig. 2. Morphological observation of normal Hela cells (4400 ).
Fig. 4. Morphological observation of anoxic Hela cells (10,000 ).
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Table 1 Comet tail lengths in Hela cells acutely treated with different doses of RSP. Group (mg/L)
Comet tail length (mm)
Overall length (mm)
Control 20 80 320 640
0.5170.08 23.9370.45 32.9670.39 37.6370.62 47.1870.65
0.8570.23 34.2870.52 53.1870.47 63.5370.67 82.1770.55
Po0.05. Po0.01.
Fig. 5. Inhibition of cell cycle progress in Hela cells by treatment with different doses of RSP for 4 days. (A) 20 mg/L RSP; (B) 80 mg/L RSP; (C) 320 mg/L RSP; (D) 640 mg/L RSP. Cells were fixed in ethanol and stained with PI, and then cell-cycle phase distribution was analyzed by flow cytometry.
Table 2 Effect of RSP on cell cycle distribution in Hela cells (n ¼ 3). Group
G0/G1 (%)
S (%)
G2/M (%)
Control 20 mg/L 80 mg/L 320 mg/L 640 mg/L
55.2671.85 54.6572.31 53.4972.33 54.0272.41 43.2472.53
38.7571.66 37.2472.35 40.8972.41 43.1572.36 54.1772.75
6.0070.63 8.1171.55 5.6270.59 2.8370.43 2.5970.35
Po0.05. Po0.01.
G2/M phases were calculated using CellQuest and Modfit software and are indicated in the Table 2. Flow cytometry DNA analysis of the treated cultures revealed that RSP induced concentrationdependent cell cycle arrest in the S phase, which was accompanied by the G0/G1 phase cells were markedly decreased from 55.26% to 43.24%. A significant accumulation of cells in the S phase (38.75–54.17%) was observed at various RSP doses. This confirmed that RSP blocked DNA replication in the S phase of the cell cycle. A sub-G1 population, normally associated with apoptotic cells, appeared in treated cells and not controls.
Discussion Recently, it has been found that peptides derived from bacterial, plant, and animal sources possess significant biological activities. These bio-active compounds are known to range from simple dipeptides to complex linear or cyclic structured peptides (Gill et al., 1996). Rapeseed meal contains 34–50% protein after extracting the oil, the amino acid composition of which is well-balanced in regard to FAO requirements and is rich in sulfur-containing amino acids and lysine, generally the limiting constituent in both legumes and cereals (Xue et al., 2004). However, there has been few reports studied on rapeseed protein hydrolysate. Our lab has done some of work about rapeseed peptide. According to our lab previous experiments, rapeseed protein isolate were enzymatically digested with proteases to investigate their potential bioactivities. The degree of enzymatic hydrolysis (DH%) was different with the treated enzymes, especially, in that study, the DH of RSP (hydrolyzed by sequential treatment with alcalase and flavourzyme) increased progressively from 14.7% to 30% and the bitterness decreased by 60%, compared to the alcalase hydrolysate (Xue et al., 2009). According to our preliminary studies, rapeseed peptides are highly possible bio-active principals present in rapeseed meal (Xue et al., 2006). In this study, using human cervical cancer Hela cell lines, we have shown that the cytotoxicity of RSP on cell proliferation and on the induction of apoptosis in a cell-specific manner. In these results, we have found that RSP inhibited cell proliferation in Hela cells in a dose- and time-dependent manner. TEM was used to observe the morphological changes of Hela cells with or without treatment of RSP. After the treatment with different concentrations of RSP, the morphology of Hela cells were changed including cell shrinkage, nuclear fragmentation and chromatin condensation which were the typical characters of apoptosis cells. SCGE, or the comet assay, is a very simple, sensitive, reliable and fairly inexpensive technique (Collins et al., 1997). The governing principle behind the method originally developed by Ostling and Johanson (1984) is that the broken DNA fragments or relaxed chromatin from the cells embedded in an agarose gel are lysed and migrate away from the nucleus under the action of an electrophoretic field, subsequently forming a ‘‘comet tail’’. As reported by Mohankumar et al. (2002), DNA damage was assessed in smokers’ lymphocytes by subjecting them to the SCGE assay. DNA damage induced by nickel chloride (NiCl2) in leucocytes of Swiss albino mice was studied in vivo by SCGE (Danadevi et al., 2004). Janja et al. (2007) applied the comet assay for use in fresh liver tissue, where all cell types, metabolic activity, and tissue architecture were preserved. The comet assay used in this paper was conducted to measure DNA strand damage at various levels following RSP treatment. When treated with 640 mg/L RSP, the DNA damage appeared to be the worst since the average length of the comet tails was the greatest (47.1870.65 mm; mean7SD). Comet tail lengths showed dose-dependent increases in DNA damage with RSP doses of 0–640 mg/L. Ongoing studies are directed toward identifying the effect in vivo. Flow cytometry DNA analysis revealed that RSP induced a dose-dependent S phase cell cycle arrest with an accompaniment decrease in G0/G1 phase. This confirmed that RSP induced a block at the S boundary. A typical feature of cells undergoing apoptosis is the occurrence of sub-G1 cell population by flow cytometric analysis of DNA content. After the treatment with RSP, the sub-G1 cell population could be observed clearly in Fig. 5. In conclusion, our study demonstrated that RSP could induce apoptotic, cytotoxic effects in Hela cells. RSP may be potentially useful in anticancer treatments. Based on the findings of this study, we suggest that it is important to search for significant
Z. Xue et al. / Experimental and Toxicologic Pathology 62 (2010) 519–523
changes in expression of genes involved in the regulation of apoptosis, following RSP treatment. Additional studies are needed to establish efficacy in RSP-treated models, which would support a rationale for clinical trials in carcinoma patients. Therefore, it is expected that peptides obtained from rapeseed protein hydrolysates could be utilized as a promising tumor preventive agent.
Acknowledgment This work was supported by the National Natural Science Foundation of China (Grant no. 30800767). References Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. Molecular Biotechnology 2004;26:249–61. Collins AR, Dobson VL, Dusinska M, Kennedy G, Stetina R. The comet assay: what can it really tell us?. Mutation Research 1997;375:183–93. Danadevi K, Rozati R, Saleha B, Grover P. In vivo genotoxic effect of nickel chloride in mice leukocytes using comet assay. Food and Chemical Toxicology 2004;42:751–7. Franco EL, Schlecht NF, Saslow D. The epidemiology of cervical cancer. Cancer Journal 2003;9:348–59. Gill I, Fandino RL, Jorba X, Vulfson EN. Biologically active peptides and enzymatic approaches to their production. Enzyme and Microbial Technology 1996;18: 162–183. Hengartner MO. The biochemistry of apoptosis. Nature 2000;407:770–6. Hsu YL, Kuo PL, Lin CC. Acacetin inhibits the proliferation of Hep G2 by blocking cell cycle progression and inducing apoptosis. Biochemical Pharmacology 2004;67:823–9. Janja P, Irena H, Primoz P, Metka F, Geny MG. Detection of xenobiotic-induced DNA damage by the comet assay applied to human and rat precision-cut liver slices. Toxicology in Vitro 2007;21:1134–42.
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Lima TM, Kanunfre CC, Pompe ia C, Verlengia R, Curi R. Ranking the toxicity of fatty acids on Jurkat and Raji cells by flowcytometric analysis. Toxicology in Vitro 2002;16:741–7. Marczak ED, Usui H, Fujita H, Yang YJ, Lipkowski MY, Yoshikawa M. New antihypertensive peptides isolated from rapeseed peptides 2003; 24: 791–8. Maria MY, Justo P, Cristina M, Julio GC, Manuel A, Francisco M, et al. Rapeseed protein hydrolysates: a source of HIV protease peptide inhibitors. Food Chemistry 2004;87:387–92. Mohankumar MN, Janani S, Prabhu BK, Kumar PV, Jeevanram RK. DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay. Mutation Research 2002;520:179–87. Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochemical and Biophysical Research Communications 1984;123:291–8. Reed JC. Apoptosis-regulating proteins as targets for drug discovery. Trends in Molecular Medicine 2001;7:314–19. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 1988;175:184–91. Xue ZH, Wu MC, Luo ZY, Yan FW, Zhou ZJ. Study on the scavenging radicals effect of rapeseed peptide (RSP). Science and Technology of Food Industry 2005; 26(10):71–5. Xue ZH, Wu MC, Yin JZ, Ruan Z. Isolation and characterization of defatted Hua-Za 3 meal protein. Food Science 2004;25(4):25–8. Xue ZH, Yu WC, Liu ZW, Wu MC, Kou XH, Wang JH. Preparation and antioxidative properties of a rapeseed (Brassica napus) protein hydrolysate and three peptide fractions. Journal of Agricultural and Food Chemistry 2009;57:5287–93. Xue ZH, Yu WC, Wu MC, Liu CQ. Effect of rapeseed peptides on tumor growth and immune function in mice. Journal of the Chinese Cereals and Oils Association 2007b;22(1):73–5. Xue ZH, Yu WC, Wu MC, Zhou ZJ, Liu CQ. Preparation and effects on Angiotensin I-converting enzyme activity of rapeseed peptide (RSP) in rats. Food Science 2007a;28(2):289–92. Xue ZH, Yu WC, Yan FW, Wu MC, Zhou ZJ. Antioxidant activity of rapeseed peptide (RSP). China Oil and fats 2006;31(8):48–50. Zeng XB, Wu MC, Li XD. Effect of rapeseed peptides on tumor growth and immune function in mice. Acta Nutrimenta Sinica 2002;24(4):405–7. Zeng XB, Wu MC, Li XD, Lin YC. Effect of rapeseed peptides on tumor cell S180 growth incubated in vitro. Acta Nutrimenta Sinica 2004;6(3):216–19.
Contents lists available at ScienceDirect
Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp
Effect of rapeseed peptide on DNA damage and apoptosis in Hela cells Zhaohui Xue a,b,1, Zhiwei Liu c,1, Moucheng Wu b, Shiwen Zhuang a, Wancong Yu d, a
School of Agriculture and Bioengineering, Tianjin University, Tianjin 300072, PR China Department of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China National Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, PR China d Department of Bioscience and Technology, Tsinghua University, Beijing 100084, PR China b c
a r t i c l e in fo
abstract
Article history: Received 16 December 2008 Accepted 30 June 2009
Rapeseed peptide (RSP), obtained by hydrolyzing rapeseed protein, has anticancer activity. In this study, the effects of RSP on proliferation rate, morphological changes, DNA damage, cell cycle distribution and apoptosis in human cervical carcinoma (Hela) cells were investigated. RSP treatment at a concentration of 640 mg/L for 4 days inhibited Hela cell proliferation significantly, as determined by the MTT assay. We observed a dose-dependent increase in cytotoxicity induced by RSP at 20–640 mg/L. After 4 days of 320 mg/L RSP treatment, typical apoptotic changes were observed by transmission electron microscopy (TEM). Using the comet assay, we found dramatic comet tails, indicating DNA damage by RSP (20–640 mg/L). Moreover, RSP treatment caused inhibition of Hela cell growth, with cycle arrest in the S phase and apoptosis induction. Taken together, the results suggested that rapeseed peptide could be a potential antitumor compound with an apoptotic mode of action. & 2009 Elsevier GmbH. All rights reserved.
Keywords: Apoptosis Rapeseed peptide (RSP) Hela cells Cytotoxicity Comet assay Cell cycle
Introduction Rapeseed peptide (RSP) belongs to a group of polypeptides that are obtained following hydrolysis of the rapeseed protein and have anticancer, immunomodulatory, antioxidant, antihypertensive and HIV protease inhibitory effects (Zeng et al., 2002; Xue et al., 2005, 2006, 2007a, 2007b; Maria et al., 2004; Marczak et al., 2003). In vitro and in vivo studies have demonstrated that RSP is an effective anticancer compound. In vivo, RSP can dosedependently decrease tumor weight and increase the amount of splenocytes, proliferation of activated T cells, NK activity and TNF levels in S180-bearing mice (Zeng et al., 2002; Xue et al., 2007b). In vitro, 20 mg/L RSP inhibited the growth of S180 cells and decreased membrane fluidity (Zeng et al., 2004). Apoptosis is the process of programmed cell death, and is tightly controlled. It plays important roles in many normal processes, ranging from fetal development to adult tissue homeostasis (Reed, 2001). During apoptosis, the nucleus and cytoplasm condense to produce membrane-bound apoptotic bodies that are phagocytosed by macrophages or adjacent cells. Emerging evidence has demonstrated that the anticancer activities of certain chemotherapeutic agents involve the induction of apoptosis, which is regarded as the most effective method of managing cancer (Hengartner, 2000; Hsu et al., 2004).
Cervical carcinoma is one of the most common neoplastic diseases affecting women, second only to breast cancer, with almost half a million new cases diagnosed annually, worldwide (Franco et al., 2003). Therefore, the development of chemotherapeutic/chemopreventive agents against cervical carcinoma is vital to reducing the prevalence and mortality of this disease. The main aim of this paper was to study the cytotoxicity of RSP. To do this, we investigated the effects of RSP treatment on DNA damage, the stage of cell cycle arrest, and apoptosis in human cervical carcinoma (Hela) cells.
Materials and methods RPMI-1640, fetal calf serum (FCS), penicillin G, streptomycin and trypsinase were obtained from Invitrogen (Carlsbad, CA). Dimethyl sulphoxide (DMSO), propidium iodide (PI), ethidium bromide (EB), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), Fluorouracil (5-FU) (Jinyao company, Tianjin, China), phosphate-buffered saline (PBS, pH 7.2–7.6) and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO). Normal melting point agarose (NMA) and low melting point agarose (LMA) were purchased from the Huamei Company (Beijing, China). Cell culture
Corresponding author. Tel.: +86 10 62773664.
E-mail address: [email protected] (W. Yu). These authors contributed equally to this work.
1
0940-2993/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.06.013
Hela cells were supplied by the Chinese Center for Disease Control and Prevention (China CDC). They were maintained as a
520
Z. Xue et al. / Experimental and Toxicologic Pathology 62 (2010) 519–523
monolayer culture at 37 1C and 5% CO2 in RPMI-1640 supplemented with 10% FCS, 10 U/mL penicillin, 10 mg/mL streptomycin, 200 mg/L L-Glutamine (Sigma) and 5 ml/L insulin (Sigma). Preparation of RSP RSP (rapeseed peptide), hydrolyzed from rapeseed protein, was isolated with alcalase and flavourzyme in our laboratory. RSP was prepared as a stock solution of 3200 mg/L in basal medium RPMI1640 and kept at 20 1C. The stock solution was diluted to appropriate final concentrations with the culture medium prior to use in all experiments. Control cultures received the same volume of RPMI-1640. MTT cytotoxicity assay The cytotoxicity induced by RSP was measured by the MTT assay. Briefly, Hela cells were plated in 96-well culture plates (5.0 104 cells per well). After a 24 h incubation period, the cells were treated with RSP (20–640 mg/L) for 2, 4, and 6 days. 160 mg/L Fluorouracil (5-FU) was selected as positive control. The medium was replaced every other day. Dye solution (10 mL), specific for the MTT assay, was added to each well for an additional 4 h at 37 1C. After adding the DMSO stop solution (100 mL/well), the absorbance (A) at 620 nm was measured by an ELISA reader (Bio-TEK, USA). The percent viability of the treated Hela cells was calculated as follows: (A620 nm) sample/(A620 nm) control 100%. Morphological observation by TEM Hela cells were cultured with basal medium and medium containing 320 mg/L RSP for 4 days. Then, the cells were examined by TEM (transmission electron microscope; Philips EM 208 s) in the following procedure: the monolayer cells were detached by trypsinization and washed twice with PBS. The cell pellets were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde at 4 1C for 2 h. The cells were then washed three times with cacodylic acid sodium salt buffer (pH 7.2) and postfixed in 1% osmic acid at 4 1C for an additional 2 h. After dehydration with gradient ethanol and 1,2-epoxypropane (twice for 15 min each). The samples were first placed in 1,2-epoxypropane and resin (V/V, 1:1) for 1 h, followed by 1,2-epoxypropane and resin (V/V, 1:4) for another hour, and then in 100% resin at room temperature for 2 h. Finally, Epon812 resin was solidified at 35 1C for 16 h, 45 1C for 8 h, 55 1C for 14 h and 60 1C for 48 h. Ultrathin sections were stained with uranyl acetate and lead citrate for examination by TEM. The control groups, normal and anoxic Hela cells, were similarly prepared. Comet assay The comet assay, also called the single cell gel electrophoresis (SCGE), is a simple, rapid and sensitive method for measuring DNA strand breaks at the level of individual eukaryotic cells (Collins, 2004). The comet assay was performed following the alkaline procedure described by Singh et al. (1988), with minor modifications. Slides were cleaned with 100% ethanol and air-dried. Two solutions containing 0.5% normal melting agarose (NMA) and 0.5% low melting agarose (LMA) were made. Briefly, 120 mL of 0.5% NMA at 45 1C was transferred onto a pre-cleaned slide, spread evenly, and placed at room temperature, allowing the agarose to solidify. Afterwards, 75 mL of 0.5% LMA at 37 1C with 5 mL of cell suspension (3 106 cells/mL) were applied and covered with a coverslip. Subsequently, slides were kept at 4 1C for up to 5 min, to allow the agarose to solidify. The coverslip was then carefully
removed. Finally, a third layer, consisting of 80 mL of 0.5% at 37 1C, LMA was added. After solidification, slides were immersed in icecold, freshly prepared lysis solution (1% sodium sarcocinate, 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10.0, 1% Triton X-100, 10% DMSO) at 4 1C for at least an hour. Then, slides were placed in a horizontal electrophoresis device filled with freshly prepared electrophoresis buffer (1 mM Na2EDTA, 300 mM NaOH) and subjected to 25 V and 300 mA for 15 min. Afterwards, slides were neutralized with 0.4 M Tris–HCl (pH 7.5), stained with 20 mL of ethidium bromide (EB, 10 mg/mL) and kept in a moist chamber in the dark at 4 1C until analysis. All the above steps were carried out under a red light to prevent any additional DNA damage. EB-stained nucleotides were viewed with a fluorescence microscope at 200 magnification using blue light excitation. The comet’s tail length was measured from the trailing edge of the nucleus to the leading edge of the tail using a calibrated slide in the ocular of the microscope. The absolute length in micrometers was calculated using a calibrated stage micrometer. A minimum of 50 cells were counted for each sample. Analysis of cell-cycle phase distribution by flow cytometry PI is a highly water soluble, fluorescent compound that cannot pass through intact membranes and is generally excluded from viable cells. It binds to DNA by intercalating between the bases in double stranded nucleic acids of exposed nuclei (Lima et al., 2002). After fixed and permeabilized cells were stained with PI, the relative fluorescence intensity of PI was measured by flow cytometry as an indirect measure of the cellular DNA content. After 4 days of culture with medium containing 20, 80, 320, 640 mg/L RSP, Hela cells were collected by trypsinization, washed with PBS and fixed in ice-cold 70% ethanol for at least 24 h. The cells were washed twice with PBS by centrifuging at 1000 rpm for 10 min. The cells were resuspended in 3 ml of PBS for 5 min. Then, the cells were stained with 1 ml PI (0.1 mg/mL with 0.1% TritonX100) and incubated in the dark for 30 min. The samples were analyzed by flow cytometry (FACSCalibur, BD, USA). Cells with a lower DNA content than that of the G1 phase of the cell cycle were considered as hypodiploid cells (sub-G1 phase). The percentage of cells in a particular phase was analyzed using the CellQuest and Modfit software (Beckman, USA). A flow cytometer equipped with a 15 mW argon laser and an excitation line of 488 nm was used. Fluorescence intensity of PI was detected at an emission wavelength 630 nm. Statistical analysis The results are expressed as mean7standard deviation (SD). The difference between control and RSP-treated cells was evaluated using the Student’s t-test. P values less than 0.05 were considered statistically significant.
Results MTT cytotoxicity assay Hela cells were cultured in 10% FCS-containing medium with or without RSP for 6 days. 20–640 mg/L RSP were selected according to the results of pre-experiments. Cytotoxicity was then evaluated using the MTT test. Under our experimental conditions, a dramatic decrease in proliferation was observed 4 days after RSP treatment (20, 80, 320 and 640 mg/L) (Fig. 1). The greatest inhibition was seen after 4 days of treatment with 640 mg/L (40%, Po0.05).
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Morphological observation by TEM As observed by TEM, the karyon was bigger and smoother in normal Hela cells (Fig. 2) than in cells treated with 320 mg/L RSP (Fig. 3). Many ‘‘apoptotic bodies’’ were observed in the RSP-treated cells. The compact masses of chromatin aggregated along the nuclear membrane. Round, compact granular masses appeared near the center of the nucleus and there was a reduction in nuclear volume. At the same time, the cytoplasm displayed condensation. Some of the nuclei degenerated into discrete spherical or ovoid fragments of highly condensed chromatin. Compared with normal (Fig. 2) and anoxic Hela cells (Fig. 4), RSP-treated cells exhibited dramatic morphological changes, characteristic of apoptotic cells. These results demonstrated that RSP induced apoptosis in Hela cells.
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damage, was measured using an ocular micrometer. The results obtained are summarized in Table 1. Typically, normal Hela cells showed comets with bright heads and small tails, while 640 mg/L RSP-treated cells exhibited comets with small heads and long diffuse tails, showing the increase of the comet tail length from 0.51 to 47.18 mm. Comets with intermediate characteristics were observed in Hela cells treated with 80, 320 and 640 mg/L RSP. A gradual increase in the tail length of comets occurred with increasing concentrations of RSP, indicating increasing DNA damage.
Analysis of cell-cycle phase distribution by flow cytometry Cell-cycle phase distribution was analyzed by flow cytometry with PI staining (Fig. 5). The percentages of cells in G0/G1, S and
Comet assay Undamaged cells had intact nuclei without tails, unlike damaged cells. The length of the comet’s tail, representing DNA
120% 0mg/L 20mg/L 80mg/L
Proliferation
100%
320mg/L
80%
640mg/L
60%
40% 0
2
4
6
8
Time (days) Fig. 1. Effect of RSP on Hela cell growth. After 24 h of adherence, Hela cells were cultured in 10% FCS medium and treated with RSP (20–640 mg/L) for 2–6 days. Results are presented as the proliferation ratio of RSP-treated Hela cells. Values were expressed as means7SD of six experiments (n ¼ 6) (P-value relative to control group: Po0.05).
Fig. 3. Morphological observation of Hela cells incubated in medium containing 320 mg/L RSP (8300 ).
Fig. 2. Morphological observation of normal Hela cells (4400 ).
Fig. 4. Morphological observation of anoxic Hela cells (10,000 ).
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Table 1 Comet tail lengths in Hela cells acutely treated with different doses of RSP. Group (mg/L)
Comet tail length (mm)
Overall length (mm)
Control 20 80 320 640
0.5170.08 23.9370.45 32.9670.39 37.6370.62 47.1870.65
0.8570.23 34.2870.52 53.1870.47 63.5370.67 82.1770.55
Po0.05. Po0.01.
Fig. 5. Inhibition of cell cycle progress in Hela cells by treatment with different doses of RSP for 4 days. (A) 20 mg/L RSP; (B) 80 mg/L RSP; (C) 320 mg/L RSP; (D) 640 mg/L RSP. Cells were fixed in ethanol and stained with PI, and then cell-cycle phase distribution was analyzed by flow cytometry.
Table 2 Effect of RSP on cell cycle distribution in Hela cells (n ¼ 3). Group
G0/G1 (%)
S (%)
G2/M (%)
Control 20 mg/L 80 mg/L 320 mg/L 640 mg/L
55.2671.85 54.6572.31 53.4972.33 54.0272.41 43.2472.53
38.7571.66 37.2472.35 40.8972.41 43.1572.36 54.1772.75
6.0070.63 8.1171.55 5.6270.59 2.8370.43 2.5970.35
Po0.05. Po0.01.
G2/M phases were calculated using CellQuest and Modfit software and are indicated in the Table 2. Flow cytometry DNA analysis of the treated cultures revealed that RSP induced concentrationdependent cell cycle arrest in the S phase, which was accompanied by the G0/G1 phase cells were markedly decreased from 55.26% to 43.24%. A significant accumulation of cells in the S phase (38.75–54.17%) was observed at various RSP doses. This confirmed that RSP blocked DNA replication in the S phase of the cell cycle. A sub-G1 population, normally associated with apoptotic cells, appeared in treated cells and not controls.
Discussion Recently, it has been found that peptides derived from bacterial, plant, and animal sources possess significant biological activities. These bio-active compounds are known to range from simple dipeptides to complex linear or cyclic structured peptides (Gill et al., 1996). Rapeseed meal contains 34–50% protein after extracting the oil, the amino acid composition of which is well-balanced in regard to FAO requirements and is rich in sulfur-containing amino acids and lysine, generally the limiting constituent in both legumes and cereals (Xue et al., 2004). However, there has been few reports studied on rapeseed protein hydrolysate. Our lab has done some of work about rapeseed peptide. According to our lab previous experiments, rapeseed protein isolate were enzymatically digested with proteases to investigate their potential bioactivities. The degree of enzymatic hydrolysis (DH%) was different with the treated enzymes, especially, in that study, the DH of RSP (hydrolyzed by sequential treatment with alcalase and flavourzyme) increased progressively from 14.7% to 30% and the bitterness decreased by 60%, compared to the alcalase hydrolysate (Xue et al., 2009). According to our preliminary studies, rapeseed peptides are highly possible bio-active principals present in rapeseed meal (Xue et al., 2006). In this study, using human cervical cancer Hela cell lines, we have shown that the cytotoxicity of RSP on cell proliferation and on the induction of apoptosis in a cell-specific manner. In these results, we have found that RSP inhibited cell proliferation in Hela cells in a dose- and time-dependent manner. TEM was used to observe the morphological changes of Hela cells with or without treatment of RSP. After the treatment with different concentrations of RSP, the morphology of Hela cells were changed including cell shrinkage, nuclear fragmentation and chromatin condensation which were the typical characters of apoptosis cells. SCGE, or the comet assay, is a very simple, sensitive, reliable and fairly inexpensive technique (Collins et al., 1997). The governing principle behind the method originally developed by Ostling and Johanson (1984) is that the broken DNA fragments or relaxed chromatin from the cells embedded in an agarose gel are lysed and migrate away from the nucleus under the action of an electrophoretic field, subsequently forming a ‘‘comet tail’’. As reported by Mohankumar et al. (2002), DNA damage was assessed in smokers’ lymphocytes by subjecting them to the SCGE assay. DNA damage induced by nickel chloride (NiCl2) in leucocytes of Swiss albino mice was studied in vivo by SCGE (Danadevi et al., 2004). Janja et al. (2007) applied the comet assay for use in fresh liver tissue, where all cell types, metabolic activity, and tissue architecture were preserved. The comet assay used in this paper was conducted to measure DNA strand damage at various levels following RSP treatment. When treated with 640 mg/L RSP, the DNA damage appeared to be the worst since the average length of the comet tails was the greatest (47.1870.65 mm; mean7SD). Comet tail lengths showed dose-dependent increases in DNA damage with RSP doses of 0–640 mg/L. Ongoing studies are directed toward identifying the effect in vivo. Flow cytometry DNA analysis revealed that RSP induced a dose-dependent S phase cell cycle arrest with an accompaniment decrease in G0/G1 phase. This confirmed that RSP induced a block at the S boundary. A typical feature of cells undergoing apoptosis is the occurrence of sub-G1 cell population by flow cytometric analysis of DNA content. After the treatment with RSP, the sub-G1 cell population could be observed clearly in Fig. 5. In conclusion, our study demonstrated that RSP could induce apoptotic, cytotoxic effects in Hela cells. RSP may be potentially useful in anticancer treatments. Based on the findings of this study, we suggest that it is important to search for significant
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changes in expression of genes involved in the regulation of apoptosis, following RSP treatment. Additional studies are needed to establish efficacy in RSP-treated models, which would support a rationale for clinical trials in carcinoma patients. Therefore, it is expected that peptides obtained from rapeseed protein hydrolysates could be utilized as a promising tumor preventive agent.
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