Food Chemistry 128 (2011) 909–915
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Effect of AATI, a Bowman-Birk type inhibitor from Apios americana, on proliferation of cancer cell lines Youzuo Zhang a,b,c, Cunshan Zhou a,⇑, Shunming Tang b,d, Xiaojie Yu a, Yoshiaki Kouzuma b, Masami Yonekura b,⇑ a
School of Agriculture and Food Science, Zhejiang A&F University, Lin’an 311300, PR China Laboratory of Food Molecular Functionality, College of Agriculture, Ibaraki University, 3-21-1, Chuo, Ami-machi, Ibaraki 300-0393, Japan R&D Center, Nippon Meat Packers Inc., 3-3 Midorigahara, Tsukuba, Ibaraki, Japan d Jiangsu University of Science and Technology, The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, PR China b c
a r t i c l e
i n f o
Article history: Received 21 November 2010 Received in revised form 18 February 2011 Accepted 30 March 2011 Available online 5 April 2011 Keywords: Inhibitor Cancer Cell Proliferation
a b s t r a c t In this paper, the inhibitory effects of AATI on proliferation of four cancer cell lines were investigated. Compared with SBBI, AATI showed the same or stronger inhibitory effect on the proliferation rate among the cell lines tested. The morphology of the cell treated with AATI appeared irregular and the adhesion ability was changed among cells. The nuclei of the cells presented characters of apoptosis. Furthermore, the amount of phosphatidylserine in the outer leaflet of the cell membrane was increased significantly during the treatment. The genomic DNA test suggested that inhibitory effects on the proliferation of cells by AATI was caused by induction of apoptosis. Moreover, chemical modification of arginine or lysine residues in AATI resulted not only in the partial loses of protease-inhibitory activity, but also in the inhibitory effect on cancer cell lines, suggesting that the inhibitory effects on the proliferation are strongly correlated with its protease-inhibitory activity. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Apios americana Medikus is an edible tuberous legume originating from eastern North America. Due to the fact that the tubers contain a high content of proteins and are used as functional foods, the current research was focused on the functional element in the tuber. The present studies showed that the AATI (a trypsin inhibitor purified from A. americana tubers) purified from tubers has the inhibitory effects on the proliferation of cancer cell lines by the induction of apoptosis, and has similar or stronger inhibitory activity with or than that of SBBI (Soybean Bowman-Birk inhibitor). This result supported the rationality of Apios tubers as functional foods, and AATI may serve as an alternative resource for SBBI for medical applications and Apios tubers can be used as a substitute for potato to meet the Western countries demands, which will be helpful to improve the cancer incidence by food intake methods. Epidemiological research results have shown that diets containing high amounts of soybean products contribute to low cancer incidence and mortality rates, particularly for breast, colon, and prostate cancers, which are more serious in Western countries ⇑ Corresponding authors. Tel./fax: +81 571 63741276 (C. Zhou), tel./fax: +81 29 888 8683 (M. Yonekura). E-mail addresses:
[email protected] (C. Zhou),
[email protected] (M. Yonekura). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.03.117
(Messina, Persky, Setchell, & Barnes, 1994). Protease inhibitors (PIs) were the first compounds identified from dietary origin as anticancer agents, although they maybe play an antinutritional role in the digestive tract (Thompson, 1993). PIs are widely distributed in living organisms and are rich in the plant seeds. SBBI is a kind of PIs, which anticancer effect was investigated extensively. The results indicated that SBBI is an effective antitumoural agent in vivo, and in vitro in cancer model systems. This inhibitor product is presently under human phase IIa clinical trials to assess its suitability as a therapeutic agent. To date, the SBBI type protease inhibitor have been isolated and characterised from more than 16 kinds of plant seeds of Fabaceae family, such as camaratu bean (Cratylia mollis) (Paiva, Oliva, Fritz, Coelho, & Sampaio, 2006), White lupin (Lupinus albus L.) (Scarafoni et al., 2008), tepary bean (Phaseolus acutifolius) (Garcia-Gasca, Salazar-Olivo, Mendiola-Olaya, & Blanco-Labra, 2002), rice bean (Vigna umbellata T), red kidney bean (Phaseolus vulgaris), brazilian pink bean (P. vulgaris), lima bean (Phaseolus lunatus), adzuki beans (Phaseolus angularis), chikpea (Cicer arietinum), pea (Pisum sativum), lentil (Lens culinaris), and pigeon pea (Cajanus cajan, syn. Cajanus indicus) (Losso, 2008). Some of them were investigated as alternative resources for cancer control. The Apios tubers contain various proteins and are used as foods for health to reduce the risk of illness. Therefore we tried to investigate the functional elements in the tuber. Previously, we isolated
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and characterised a trypsin inhibitor, named as AATI. AATI has inhibitory activity toward trypsin and chymotrypsin, and the biochemistry characteristics and amino acid sequence homology analysis results indicated that AATI belonged to the family of Bowman-Birk type inhibitors (Zhang, Kouzuma, Miyzji, & Yonekura, 2008). In this paper, the effects of AATI on proliferation of four cancer cell lines, including U937, K562, J774.1, and HeLa, were investigated. Compared with SBBI, AATI showed the same or stronger inhibitory effect on the proliferation rate. The inhibition of the proliferation by AATI was presented in a dose- and time-dependent manner. We found that the treatment of AATI induced apoptosis. Chemical modification of AATI showed a loss of inhibitory effect on proliferation and decrease in the inhibitory activity toward trypsin and chymotrypsin. 2. Materials and methods 2.1. Materials ATTI was prepared as previously reported (Zhang et al., 2008). Four cancer cell lines, U937 (Human leukaemic monocyte lymphoma cell line) (Ralph, Moore, & Nilsson, 1976), J774.1 (a murine macrophage cell line) (Ralph & Nakoinz, 1977), K562 (human chronic myelogenous leukaemia cell line) (Lozzio & Lozzio, 1975) and HeLa (Human epithelial carcinoma cell line) (Hanaoka & Yamada, 1971), were purchased from RIKEN Bioresource Center (Ibaraki, Japan). MEM medium, RPMI-1640 medium, NEAA, penicillin, streptomycin, and trypan blue were from GIBCO (Grand Island, NY, USA). Propidium iodide solution and Bisbenzimide H33342 were from SIGMA (St. Louis, MO, USA). Apoptosis ladder detection kit and Annexin V-fluorescein kit were from WAKO (Osaka, Japan). Benzoyl-L-arginine-p-nitroanilide hydrochloride (L-BAPA) was obtained from Peptide Institute Inc. (Osaka, Japan). All other chemicals were of analytical grade for biochemical use. 2.2. Cell culture U937 and J774.1 cells were maintained in RPMI-1640 medium with 10% foetal bovine serum, and 100 U/ml penicillin and streptomycin. K562 cells were grown in MEM medium containing 10% foetal bovine serum, 100 U/ml penicillin and streptomycin and 0.1 M NEAA. HeLa cells were maintained in MEM medium with 10% foetal bovine serum, and 100 U/ml penicillin and streptomycin. All cell lines were incubated at 37 °C under a 5% CO2 atmosphere. The cells at logarithm proliferative phase are ready for the next experiment. 2.3. Cell proliferation assay The inhibitory effects of AATI on proliferation in four cancer cells were evaluated by the trypan blue exclusion assay. In brief, 100 ll cells were seeded on 96-well plates at a density of 2 104 cells/ml in the corresponding medium with or without indicated concentrations of AATI. At the end of the incubation period, the cells were harvested. The cells were mixed with trypan blue solution, and the living cells (without dye staining inside) were counted by a hemocytometer under a microscope. The living cell percentage was calculated based on the number of living cells normalised to that of the corresponding control. 2.4. Cell morphology and adhesion Cells were seeded on 24-well plates at a density of 2 104 cells/ ml in 1 ml of corresponding medium with or without indicated
concentrations of AATI. At each of the incubation periods, the cells were observed under a microscope (Leica Dmirb). 2.5. Cell nuclei morphology Cells were seeded on 24-well plates at a density of 2 104 cells/ ml in 1 ml of corresponding medium with or without 100 lg/ml AATI and incubated for 24 h. The cells were collected and stained in 2 lg/ml Bisbenzimide H33342 solution for 30 min in the dark. The cell nuclei were observed under fluorescence microscopy (Olympus DP70, Blue filter). 2.6. Determination of phosphatidylserine amount on the outer leaflet of cell membrane The amount of phosphatidylserine in the outer leaflet of cell membrane was determined according to the protocols of Annexin V-fluorescein kit. Briefly, cells were seeded on 96-well plates at a density of 2 104 cells/ml in 100 ll of corresponding medium. AATI was added to the medium at a concentration of 100 lg/ml and the cells were incubated for 24 h. The cells were harvested by centrifugation at 220g for 20 min and washed with cold PBS solution twice. The cell pellet was resuspended in 100 ll of Annexin V-fluorescein and propidium iodide solution and incubated at 25 °C for 15 min. The fluorescence of samples was analysed using a Wallac 1420 ARVOsx with appropriate excitation (485 nm) and emission (535 nm) filters. 2.7. DNA fragmentation assay Cells were seeded on 24-well plates at a density of 2 104 cells/ ml in 1 ml of corresponding medium. AATI was added to the medium at a concentration of 100 lg/ml and cells were incubated for 24 h. Genomic DNA was extracted from cells by using a DNA laddering kit provided by WAKO Japan. Samples were analysed on 1.5% agarose minigel and visualised with ethidium bromide under ultraviolet light. 2.8. Modification of arginine or lysine residues in AATI Modification of the arginine residue in AATI was performed according to the method of Patthy (Patthy & Smith, 1975). AATI were treated with 0.05 M cyclohexanedione in 0.2 M sodium borate at pH 9.0 at a protein concentration of 5 mg/ml at 37 °C for 30 min. All concentrations were given pertain to the final reaction mixture. After reaction, the sample was dialysed against 1% acetic acid for 24 h at 4 °C and lyophilised. The Arg-modified thus obtained was designated as AATI-Arg. The succinylation of the lysine residue with succinic anhydride was performed according to the method of Ohba et al. (1998). Briefly, AATI (1 mg/ml) was dissolved in 0.5 M NaHCO3, and succinic anhydride dissolved in N,N-dimethyl-formamide (DMF) was added (final concentration of succinic anhydride and DMF were 12.5 mM and 2.5% (v/v), respectively). The solution was incubated at 20 °C for 30 min, and then dialysed against 5 mM Tris buffered saline (TBS) overnight at 4 °C and lyophilised. The Lys-modified AATI thus obtained was designated as AATI-Lys. 2.9. Trypsin inhibitory activity assay Inhibition of trypsin was assayed by the method of Shibata and Hara (Shibata, Hara, Ikenaka, & Ase, 1986). Trypsin was dissolved in 1 mM HCl containing 20 mM CaCl2 at a concentration of 0.02 mg/ml. Then 100 ll of the trypsin solution was incubated with 200 ll of the inhibitor solution in 50 mM Tris–HCl buffer, pH 7.5, at 37 °C for 10 min, and then 1 ml of 0.5 mM L-BAPA
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2.10. Statistical analysis
120
Living cell percentage (%)
substrate solution in 50 mM Tris–HCl buffer (pH 7.5) was added. After incubation at 37 °C for another 10 min, 200 ll of 30% acetic acid was added to end the enzyme reaction, and the absorbance at 410 nm was measured on a spectrophotometer.
40 20 48
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Incubation time (h)
120 Living cell percentage (%)
The inhibitory effects on the proliferation for U937, K562, J774.1, and HeLa cell lines with AATI treatment of different concentrations are presented in Fig. 1. Compared with the control, the number of living cells treated with AATI decreased significantly among the four cancer cell lines. Furthermore, the increase of the dose of ATTI, living cell percentage decreased at 24, 48, and 72 h post treatment (hpt). For U937, J774.1, and HeLa cells, the increased incubation time resulted in a decrease of living cell percentage, while it resulted in an increase of living cell percentage for K562 cells. These results indicated that the inhibitory action of AATI was in a dose- and time-dependent manner. Compared with the result of treatment with SBBI at 100 lg/ml, living cell percentage from AATI treatment at the same concentrations were similar in U937, K562 and HeLa cells, and was highest in J774.1 cells.
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K562
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3.1. Inhibitory effects of AATI on the proliferation of four cancer cell lines
80
0
Data were analysed by one-way ANOVA followed by Student’s t-test. p Values of 0.05 or less were considered significant. 3. Results and discussion
U937
100
J774.1
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3.2. Effects on the morphology and adhesion ability of cell
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Incubation time (h)
3.3. Induction of apoptosis by AATI After treated with 100 lg/ml AATI for 24 h, the cell nuclei were stained with 2 lg/ml Bisbenzimide H33342 solution (Fig. 4). The volume of cell nuclei in the treatment was increased and the appearance of cell nuclei seem rippled and creased, which suggested that the apoptosis was proceeding in the cells. Therefore, more detailed experiments on apoptosis were carried out. When the cells were stained with the mixed solution of Annexin V-fluorescein and propidium iodide, it was found that some of K562 and HeLa cells were at stage I (green), some of U937 and J774.1 cells were at stage II (yellow), or at stage III (red) (Fig. 5), which showed that the cells were in different stages of the apoptosis process. Next, the amount of phosphatidylserine in the outer leaflet of cell membrane was determined (Fig. 6). Compared with the control, the treatment by AATI resulted in the high fluorescence intensity from 2 hpt. Fluorescence intensity of K562, HeLa, J774.1, and U937 cells was 8417 ± 504, 7920 ± 354, 8193 ± 402, 6574 ± 234, respectively. Since the levels were similar from 2 to 24 hpt, it was suggested that the effect of AATI on the cell membrane appeared rapidly and continuously. Then the DNA fragmentation was analysed in the cells treated with AATI (Fig. 7). Compared with the control, the treatment led
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Living cell percentage (%)
Change of the morphology of U937, K562, J774.1, and HeLa cells with AATI treatment of 100 lg/ml at 24 hpt is shown in Fig. 2. Compared with the control, the treated cell’s volume was relatively small and the protuberance and disfiguration appeared in the cell membrane. At 96 hpt, the treated cells were easy to pile up together (Fig. 3), which suggested that the treatment of AATI increased the adhesion ability of the four tumour cells.
Hela
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Incubation time (h) Control
AATI 12.5µg/ml
AATI 25µg/ml
AATI 50µg/ml
AATI 100µg/ml
AATI 200µg/ml
AATI 400µg/ml
BBI 100µg/ml
Fig. 1. Effect of AATI on the proliferation of U937, K562, J774.1, and HeLa cells. Cells were treated with AATI at concentrations of 12.5, 25, 50, 100, 200, and 400 lg/ml, respectively, or soybean BBI (100 lg/ml) for 24, 48, and 72 h. Living cell percentage were estimated by the method as described in Section 2. Control, vehicle treatment. Each value indicates the means from three samples; vertical lines indicate SE. ⁄ Significant difference (p < 0.05) from control by Student’s t-test.
to degradation of the genomic DNA of the cell and the small size of DNA fragments were detected as smear bands. 3.4. Effects of chemical modification in AATI on the proliferation of cells To elucidate the relationship between the protease inhibitory activity of AATI and its inhibition effect on the cell proliferation, two inhibitor reactive sites (Lys62 and Arg88) were modified chemically and the activities of the modified AATI were investi-
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+ AATI
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U937
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Fig. 2. Morphological changes of the cells treated with 100 lg/ml AATI for 24 h. Cells morphology was observed by a phase contrast inverted microscope (100). (A–D) Vehicle treatment; (a–d) treatment with AATI at a concentration of 100 lg/m for 24 h.
-AATI
Fig. 4. Morphological changes of nuclei of cells treated with 100 lg/ml AATI for 24 h. Cells were treated with AATI at a concentration of 100 lg/ml for 24 h. Cells were stained with Bisbenzimide H33342 solution and the observation method was as described in Section 2. AATI column, vehicle treatment; +AATI column treatment with AATI at concentration of 100 lg/ml for 24 h.
+ AATI
- AATI
U937
U937
K562
K562
J774.1
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Fig. 3. Effect of AATI on the cell adhesion of U937, K562, J774.1 and HeLa cells. Cells morphology was observed by a LEICA DMIRB microscope (100). AATI column, vehicle treatment; +AATI column treatment with AATI at a concentration of 100 lg/ ml for 96 h.
gated. When Lys or Arg residues in AATI were modified, the inhibitory activity on the trypsin and chymotrypsin were significantly decreased (data not shown). The inhibitory effects of the modified ones on the proliferation of cancer cells was nearly diminished at 24 hpt (Fig. 8). This result demonstrated clearly that the inhibitory effects of ATTI on the proliferation of cancer cells are positively related with its inhibitory activity toward trypsin and chymotrypsin.
Fig. 5. Detection of phosphatidylserine on the outer leaflet of cells membrane treated with 100 lg/ml AATI for 24 h. Cells were treated with AATI at a concentration of 100 lg/ml for 24 h. Cells were stained with Annexin V-fluorescein and propidium iodide solution and the observation method was as described in Section 2. AATI column, vehicle treatment; +AATI column, treatment with AATI at concentration of 100 lg/ml for 24 h.
SBBI use as a cancer chemopreventive agent has been well-documented and has a possible future in the application of clinical agents (Losso, 2008). Research has started to elucidate the underlying mechanisms by which BBI exerts anticarcinogenetic effects and several pathways have been proposed. The observed effects can be classified as several basic types: signal transduction pathways, DNA repair processes, and inhibitor for the nuclear proteases and the proteasome, which degraded the extracellular matrix etc.
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Fig. 6. Changes of the fluorescence intensity of the outer leaflet of cell membrane treated with AATI. Cells were treated with AATI(+) at a concentration of 100 lg/ml for 2, 4, 8, 12, and 24 h. Control( ), vehicle treatment. Fluorescence intensity was estimated by the method as described in Section 2. Each value indicates the means from three samples; vertical lines indicate SE. ⁄Significant difference (p < 0.05) from control by Student’s t-test.
+
-
+
-
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HeLa
)
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J774.1
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U937 M
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Fig. 7. Agarose gel electrophoresis of DNAs extracted from cells treated with AATI of 100 lg/ml for 24 h. Cells were treated with AATI(+) at a concentration of 100 lg/ ml for 24 h. Control( ), vehicle treatment. DNA extracts prepared and analysed by the method as described in Section 2. M, ladder marker.
(Chen, Huang, Lin-shiau, & Lin, 2005; Dittmann, Knaus-Dittmann, Mayer, & Rodemann, 2001; Stoner, Morse, & Kelloff, 1997). It is reported that the inhibitory effect of serine proteinase inhibitor on the proteasome triggered cell apoptosis (Chen, Huang, Lin-shiau, and Lin, 2005; Hara et al., 1996). The present studies results indicated that the inhibitory effect of AATI on the proliferation of cancer cells came from the cell apoptosis. We deduced that AATI maybe also the inhibitor for proteasome, which resulted in the apoptosis in the four cancer cell lines tested. The inhibitory effects on the proliferation of four cancer cell lines are different maybe due to the fact that the cancer cell line originated from different sources. Garcia-Gasca and coworkers reported that protease inhibitor activity (TPIF), a kind of BBI, has an inhibitory effect on the proliferation of 3T3/v-mos cell lines, while no effect on the HeLa and normal 3T3 cells (Garcia-Gasca et al., 2002). In the present research, AATI modified in Lys or Arg residues lost the inhibitory effect on the cancer’s proliferation. Similar results have also been reported in work by Ohba, which describes that the chemical modified Lys in EBI, a kind of BBI from Erythrina variegata, lost the cytotoxicity on Molt 4 cells (Ohba et al., 1998). The anticarcinogenic activity of PIs has been directly related to their ability to inhibit chymotrypsin like proteases. PI amino acids of the reactive sites of AATI are Arg88 and Lys62, which means that AATI is a trypsin inhibitor. However, AATI has chymotrypsin
inhibitory activity, suggesting that the activity of AATI contributed the proliferation on cancer cells. It is reported that mammary tumours were deficient in gap junction and cells derived from mammary tumour do not exhibit gap junction-mediated cell-cell communication (Lee, Tomasetto, Paul, Keyomarsi, & Sager, 1992; Wilgenbus, Kirkpatrick, Knuechel, Willecke, & Traub, 1992). Connexins are proteins found in gap junctions, which served as potential anti-oncogenic targets for cancer chemoprevention and chemotherapy (Pointis, Fiorini, Gilleron, Carette, & Segretain, 2007; Sawey, 2001). Some of connexins were regulated by the SBBI administered (Saito et al., 2007; Sawey, 2001). The SBBI-treatment increased the amount of connexin 43 in M5076 and U2OS cancer cells, which is directly associated with the suppression of sarcoma development (Saito et al., 2007; Suzuki et al., 2005). The increased connexin maybe resulted in the adhesion ability among cells. In the present study the treatment of AATI increased the adhesion ability of four cancer cells. The same effect of tepary bean seeds with protease inhibitor activity (TPIF) on 3T3/ v-mos cell lines was reported (Garcia-Gasca et al., 2002). Further experiments are needed to elucidate whether the connexin is involved in the inhibitory effect of AATI on the proliferation of cancer cells. SBBI has the characteristics of selective inhibitory effect on the cancer cells, other than the normal cells (Dittmann et al., 2005; Tang et al., 2009). Whether the AATI has the same characteristics need further experiments to clarify. The incidence and mortality rates for several cancers are higher in Western populations, which may result from their limited consumption of soy products. Krishnan found that Apios tuber may be a suitable candidate for Western populations (Krishnan, 1998). One reason is the high content of genistein in the tuber, which is also another kind of anticancer reagent. On the other hand, potato is widely consumed by the Western population. Since Apios tubers share similar characteristics of potato, perhaps it may be easier to substitute for potato without drastically altering the eating patterns of the Western population (Krishnan, 1998). A. americana has been evaluated under field conditions for its food crop potential (Hoshikawa & Juliarni, 1995). The current results that AATI in this tuber has inhibitory function on the cancer cells reinforce the above opinion, suggesting that Apios tuber can be used as a substitute for potato to meet the Western countries demands, which will be helpful to improve the cancer incidence by food intake method.
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Living cell percentage (%)
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investigated. Compared with soybean BBI, AATI showed the same or stronger inhibitory effect on the proliferation rate among the cell lines tested. The morphology of cells treated with AATI appeared irregular and the adhesion ability was changed among cells. The nuclei of the cells presented characteristics of apoptosis. Furthermore, the amount of phosphatidylserine in the outer leaflet of cell membrane increased significantly during the treatment. Agarose gel electrophoresis of the genomic DNA showed that the DNA was broken into the fragment state by treatment with AATI. These results suggested that inhibitory effects on the proliferation of cells by AATI were caused by induction of apoptosis. Moreover, chemical modification of arginine or lysine residues in AATI resulted not only in the partial loses of protease-inhibitory activity, but also in the inhibitory effect on cancer cell lines, suggesting that the inhibitory effects on the proliferation are strongly correlated with its protease-inhibitory activity.
U937
90
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30 0
24
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Incubation time (h)
Living cell percentage (%)
120
K562
90
Acknowledgements The authors are grateful for the financial support provided by National Natural Science Foundation of China (Project No. NSFC, 30940058).
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Living cell percentage (%)
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Hela
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Incubation time (h) Control
AATI
AATI-Arg
AATI-Lys
Fig. 8. Effects of AATI and the modified AATIs on the cell proliferation. Cells were treated with AATI, AATI-Arg or AATI-Lys at a concentration of 100 lg/ml. Living cell percentage were estimated by the method as described in Section 2. Control, vehicle treatment. Each value indicates the means from three samples; vertical lines indicate SE. ⁄Significant difference (p < 0.05) from control by Student’s t-test.
4. Conclusions In this paper, the inhibitory effects of AATI on proliferation of four cancer cell lines, including U937, K562, J774.1, and HeLa, were
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