Thermostable Kunitz trypsin inhibitor with cytokine inducing, antitumor and HIV-1 reverse transcriptase inhibitory activities from Korean large black soybeans

Journal of Bioscience and Bioengineering VOL. 109 No. 3, 211 – 217, 2010 www.elsevier.com/locate/jbiosc

Thermostable Kunitz trypsin inhibitor with cytokine inducing, antitumor and HIV-1 reverse transcriptase inhibitory activities from Korean large black soybeans Evandro Fei Fang, Jack Ho Wong, and Tzi Bun Ng⁎ School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Received 16 June 2009; accepted 26 August 2009 Available online 14 October 2009

A large number of trypsin inhibitors belonging to various types have been purified from different kinds of legumes. In this study, by using liquid chromatography, a Kunitz type trypsin inhibitor (KBTI) with a molecular weight of 20107.645 Da was purified from Korean large black soybeans. KBTI reduced the proteolytic activities of trypsin and α-chymotrypsin with the activity of ∼ 8520 BAEE units/mg and ∼ 24 BTEE units/mg, respectively. It showed high thermal stability (0–100 °C) as well as stability over a large range of pH values (pH 3–11). Furthermore, KBTI inhibited HIV-1 reverse transcriptase activity with an IC50 value of 0.71 μM and induced the release of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-2 and interferon-γ at the mRNA level. KBTI exerted weak antiproliferative activity toward CNE-2 and HNE-2 nasopharyngeal cancer cells, MCF-7 breast cancer cells, and Hep G2 hepatoma cells. KBTI was destitute of mitogenic, ribonuclease and antifungal activities. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Korean large black soybean; Glycine max; Seeds; Purification; Trypsin inhibitor; Biological properties]

There has been a long history of cultivation of soybeans in China, South Korea and other Asian countries. They now serve as important staples in the cuisines of these areas. Soybeans have been recommended as health food as they manifest antioxidant capacities (1, 2), reduce the risk of cardiovascular diseases (3, 4) and obesity (5, 6), modify cancer incidence and inhibit tumor cell proliferation (7-9), and even relieve menopause-related symptoms (10). However, the mechanisms of such health-promoting functions at the protein level have not been fully elucidated. The protease family is composed of subfamilies of serine, cysteine, metallo, aspartic and threonine proteases (11). These proteases and the corresponding inhibitors are widely distributed in plant seeds, particularly in legumes (12). An imbalance between proteases and their inhibitors may trigger a series of abnormal functions resulting in tumor growth and metastasis (11, 13). Besides sustaining intravital stability, protease inhibitors with anti-HIV (14, 15), anti-proliferative (14), antiinsect (16), and anti-fungal activities (17) have been reported. In this paper we report the isolation of a Kunitz type trypsin inhibitor from the seeds of Korean black soybean, a new cultivar of Glycine max. Their biochemical and functional properties were described including pH and thermal stability, anti-HIV-1 reverse transcriptase, antitumor and cytokine inducing abilities. To the best of our knowledge, this is the first report of a Kunitz type trypsin inhibitor of legume origin with anti-proliferative activity on nasopharyngeal carcinoma cells.

⁎ Corresponding author. Tel.: +852 26096872; fax: +852 26035123. E-mail address: [email protected] (T.B. Ng).

MATERIALS AND METHODS Reagents and tumor cell lines Korean large black soybeans (a cultivar of Glycine max) were purchased from a local market and identified by Professor Shiu-Ying Hu, Honorary Professor of Chinese Medicine, The Chinese University of Hong Kong (CUHK). Q-Sepharose™ (Fast Flow), Blue-Sepharose™ (6 Fast Flow), Mono Q 5/50 GL, Superdex 200 10/300 GL and Superdex 75 10/300 GL were obtained from GE Healthcare. Bovine pancreatic trypsin (3631 usp units/mg power), α-chymotrypsin (50 usp units/mg power) and soybean trypsin inhibitor (8657 usp units/mg power) were from USB Corporation (Cleveland). Reverse transcriptase PCR kit was purchased from GeneAmp® (New Jersey). BALB/c mice were provided by the Laboratory Animal Services Center of CUHK and handled following the appropriate ethical recommendations. The human nasopharyngeal carcinoma cell lines CNE-2 and HNE-2 were bought from Sun Yat-sen University of Medical Sciences, Guangzhou, China. Hep G2 and MCF-7 cell lines were purchased from ATCC. All others used were from Sigma (USA) or as mentioned. Purification of Korean bean trypsin inhibitor First, 400 g dried seeds were soaked in deionized water at 4 °C overnight and then extracted thoroughly using a blender (5 ml deionized water/g sample) followed by centrifugation at 20,000 × g at 4 °C for 40 min. The supernatant was filtered through filter paper and then applied to a Q-Sepharose column (18 cm × 5 cm) previously equilibrated with 20 mM NH4HCO3 buffer (pH 9.4). The column was washed with the equilibration buffer until OD280 reached the baseline and the bound fraction was eluted with 1 M NaCl in the same buffer. The eluted bound fraction (Peak Q-II) was dialyzed exhaustively against deionized water at 4 °C. After adjusting to 20 mM Tris–HCl (pH 7.6) buffer, it was applied to a Blue-Sepharose column (18 cm × 5 cm) which had been pre-equilibrated with 20 mM Tris–HCl (pH 7.6) buffer and was then washed with the same equilibration buffer. This Blue-Sepharose unadsorbed fraction (Peak B-I) from Blue-Sepharose exhibited trypsin inhibitory activity. After exhaustive dialysis against deionized water at 4 °C, lyophilization and then adjusting to 20 mM NH4HCO3 buffer, Peak B-I was applied to a Mono Q 5/50 GL column and eluted with a series of linear concentration gradients of NaCl (0–0.2, 0.2–0.6, 0.6–1 M). Active fractions were pooled, dialyzed, lyophilized and chromatographed on a Superdex 75 10/300 GL column in 200 mM NH4HCO3 buffer (pH 9.4). The purified trypsin inhibitor was dialyzed extensively, freeze-dried and stored at 4 °C for further research.

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TABLE 1. Primers of GAPDH and cytokines used in this study. Gene Name

Primers

Sequence

Product size (bp)

GAPDH

Upper Lower Upper Lower Upper Lower Upper Lower Upper Lower

5′ ACCACAGTCCATGCCATCAC 3′ 5′ TCCACCACCCTGTTGCTGTA’3 5′TCCCCAAAGGGATGAGAAGTTC3′ 5′TCATACCAGGGTTTGAGCTCAG3′ 5′AAACAGATGAAGTGCTCCTTCAGG 3′ 5′TGGAGAACACCACTTGTTGCTCCA 3′ 5′ TTGATGGACCTACAGGAGCTCCTGAGCA 3′ 5′ AGAGAGCCTTATGTGTTGTAAGCAGGAGG 3′ 5′AGGAACTGGCAAAAGGATGGTG3′ 5′GTGCTGGCAGAATTATTCTTATTG3′

452

TNF-α IL-1β Il-2 INF-γ

25 μL trypsin (1 mg/ml in 50 mM Tris–HCl containing 0.2 M CaCl2, pH 7.6) for 5 min at 37 °C. Then residual trypsin activity was measured by adding 1.45 ml 0.25 mM N-αbenzoyl-L-arginine ethyl ester (BAEE) as substrate. After immediately mixing by inversion the increase in A253 was recorded for 5 min. Reactions without addition test samples were used as positive control. Calculation:

411 306 209 353

Homogenecity and molecular mass determination Homogenecity of the purified protein was ascertained by molecular exclusion chromatography (2 mg/ml in 200 mM NH4HCO3 buffer) on a Superdex 200 10/300 GL column equilibrated and eluted with the same buffer. For molecular mass determination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12% polyacrylamide gel) was carried out as mentioned before (18) under reducing and non-reducing conditions, followed by staining with 0.1% Coomassie Brilliant Blue G. The low molecular weight SDS calibration kit from GE Healthcare (19) used is a mixture of six proteins including phosphorylase b (97.0 kDa), albumin (66.0 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa) and α-lactalbumin (14.4 kDa). In addition, molecular mass was also determined by mass spectrometric analysis. It was carried out on a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) at a trace concentration of 5 μM (20). Determination of N-Terminal amino acid sequence N-terminal amino acid sequence analysis was performed using an HP 1000A Edman degradation unit and an HP 1000 HPLC system after SDS-PAGE and membrane transferring procedures (21). Determination of trypsin inhibitory activity The assay was done by using a spectrophotometric method with some modifications (22). Firstly, 25 μL of a serial concentration (7.5 μg/ml to 1 mg/ml) of purified trypsin inhibitor was incubated with

Activity of trypsin inhibitor ðBAEE units=mgÞ
¼ DA253=min Positive−DA253=min Test =½ð0:001Þð0:025ÞðsampleÞ Where (0.001) = the change in A253/min per unit of trypsin at pH 7.6 and 25 °C in a 1.5 ml reaction mix; (0.025) or (sample) = solid (mg) of trypsin or sample used in the reaction. In addition, 1 unit of trypsin inhibitor activity is the amount of inhibitor which reduces the trypsin activity by 1 BAEE-U. 1 BAEE-U (1 trypsin unit) is the amount of enzyme which increases the absorbance at 253 nm by 0.001/min with BAEE as substrate at pH 7.6 and 25 °C. Chymotrypsin inhibitory activity was done by the same assay, but using Nbenzoyl-L-tyrosine ethyl ester (BTEE) as substrate. One chymotrypsin unit hydrolyzes 1.0 μmol of BTEE per minute at pH 7.6 and 25 °C. In addition, in order to show a dosedependent relationship, another assay was conducted using 1% casein as substrate (23). Soybean trypsin inhibitor from USB Corporation was used as positive control. Effect of the reducing agent dithiothreitol (DTT) (Sigma) on stability of the purified trypsin inhibitor was done following the method of Ramos et al. (16). The trypsin inhibitor (4 mg/ml) was incubated with a serial concentration (1, 10, 100 mM) of DTT for 30 min at 37 °C and then the reaction was terminated by adding iodoacetamide at twice the amount of each DTT concentration. The residual trypsin inhibitory activity was measured as described above. Soybean trypsin inhibitor from USB Corporation (19) was used as control. Temperature and pH stability A 20 μL aliquot of a 1 mg/ml solution of the isolated trypsin inhibitor in 20 mM Tris–HCl buffer (pH 7.6) was exposed to 0–100 °C by using a PCR machine or buffers at pH 2–12 for 0.5 h and then cooled to 0 °C before testing for residual activity as mentioned above. Remaining trypsin inhibitory activity was calculated as percent of the value at 20 °C or pH 7 (16, 24). Assay of HIV-1 reverse transcriptase (HIV-1-RT) inhibitory activity The assay was done using an HIV-1-RT (recombinant) ELISA kit following the instructions of the manufacturer (Boehringer Mannheim, Germany). The assay is based on the mechanism

FIG. 1. Purification of Korean black soybean trypsin inhibitor by chromatography. (A) The crude extract was first applied to a Q-Sepharose column and (B) the bound fraction Q-II was loaded on Blue-Sepharose to yield an unadsorbed fraction (B-I) with trypsin inhibitory activity. (C) Fraction B-I was applied to a Mono Q 5/50 GL column and (D) the MQ-I peak was subsequently loaded on a Superdex 75 10/300 GL column to yield purified trypsin inhibitor (SUP-II). The dashed line represents the concentration of NaCl employed.

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were maintained in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin. After the cells had grown to 90% confluence, the cells were trypsinized and seeded to a 96-well plate (5000 cells in 100 μL per well). After incubation for 8 h, the cells were treated with a serial dilution of the purified trypsin inhibitor at concentrations from 1.6 to 100 μM for 24 or 48 h. Two hours before the end of each incubation interval, 50 μL of a 5 mg/ml [-[4,5-dimethylthiazol-2yl]-2,5diphenyltetrazolium bromide] (MTT) solution was spiked into each well. After careful removal of the medium, 150 μl dimethyl sulfoxide was added to each well and then agitation on an orbital shaker for 15 min. The absorbance at 595 nm was measured by using a BIO-RAD Microplate Reader. Assays of other activities Other activities such as mitogenic activity, antifungal activity, and ribonuclease activity were assayed as described earlier (20, 29, 30). Statistical analysis Results were taken from three independent experiments performed in duplicate or triplicate, and data were expressed as means± standard deviation. For between-group comparisons, Student's t-test or one-way ANOVA followed by pairwise analysis with Sidak correction were used as appropriate.

RESULTS

FIG. 2. SDS-PAGE and mass spectrometric analysis results. (A). SDS-PAGE showing fractions from different stages of purification of Korean black soybean trypsin inhibitor KBTI. Lane 1, molecular weight marker proteins; lane 2, total extract; lane 3, QSepharose (bound fraction Q-II); lane 4, Blue-Sepharose (unbound fraction B-I); lane 5, Mono Q (bound fraction MQ-I); lane 6: Superdex 75 (fraction SUP-II). (B) The mass spectrometry results of KBTI showing a peak of 20107 Da. that HIV-1-RT can synthesize DNA, commencing from the template/primer hybrid poly(A) oligo (dT). The inhibitory activity of the trypsin inhibitor was calculated as percentage inhibition compared with the control without any protein added (25). Pinto bean lectin with anti HIV-1-RT activity was chosen as positive control (26). Assay of cytokine-inducing ativity Two 20–25 g BALB/c mice were sacrificed for preparation of splenocytes which were subsequently seeded into 90 mm Sterilin dishes. After treatment with the trypsin inhibitor at 100 or 20 μg/ml for 4 h (20 μg/ml Concanavalin A as positive control), total cellular RNA was isolated using TRIZOL® (Invitrogen, USA) and then adjusted to 1 μg/μL for RT-PCR. The RT and PCR reactions were conducted by using a GeneAmp® RNA PCR kit (USA) following the manufacturer's instructions as mentioned before (27). The PCR products were then resolved by electrophoresis on a 1.5% agarose gel containing ethidium bromide and visualized under UV light. The changes of mRNA level of cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interferon-gamma (INF-γ) were measured, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as control. Primers of these cytokines are shown in Table 1. Assay of antiproliferative activity on tumor cell lines The MTT test was used to monitor inhibition of cell growth (28). Briefly, CNE-2, HNE-2, MCF-7 and Hep G2 cells

Purification and molecular weight of Korean bean trypsin inhibitor The crude extract of Korean black beans was loaded on a Q-Sepharose column and the adsorbed fraction peak Q-II was subsequently applied on a Blue Sepharose column (Figs. 1A and B). The unadsorbed fraction B-I with antitryptic activity was applied to a Mono Q 5/50 GL column. The acquired MQ-I fraction was then applied to a Superdex 75 10/300 GL column with SUP-II as purified fraction (Figs. 1C and D). A 20-kDa Korean bean trypsin inhibitor, hereinafter referred to as KBTI, was obtained and its purity was ascertained by mass spectrometry (Fig. 2B). The yields of the trypsin inhibitor in different chromatographic fractions are presented in Table 2. The final yield of KBTI was about 33 mg from 400 g of beans corresponding to 19% of the total activity of the crude extract. SDS-PAGE (Fig. 2A) showed that the molecular weight of KBTI was about 20 kDa, which was in accordance with the results of mass spectrometry showing a peak with a molecular weight of 20107.645 (Fig. 2B). The N-terminal amino acid sequence is presented in Table 3. They were identical and exhibited some extent of homology to other Kunitz trypsin inhibitors. Stability and biochemcal characterization Results of Figs. 3A and B showed that KBTI was stable from 0 to 100 °C and from pH 3 to 11 and only lost stability slightly at the extreme pH values of 2 and 12. KBTI inhibited the BAEE- or BTEE-cleaving ability of both bovine trypsin and α-chymotrypsin with corresponding activities of ∼ 8520 BAEE units/mg and ∼ 24 BTEE units/mg, respectively. Figs. 4A and B showed a dose-dependent inhibition of trypsin/α-chymotrypsin by KBTI by using casein as a substrate. The value of the positive control (soybean trypsin inhibitor from USB Company) was comparable with the values reported by the manufacturer. Fig. 4C showed that DTT reduced the activity of KBTI in a dose-dependent manner from 10 mM concentration. Anti-HIV reverse transcriptase activity, cytokine-inducing activity and antiproliferative activity on tumor cell lines KBTI showed significant HIV reverse transcriptase inhibitory activity with an IC50 value of 0.71 μM (Fig. 5). The 62-kDa pinto bean lectin used as

TABLE 2. Summary of purification procedure of KBTI from 400 g of dried seeds. Fraction Crude extract Q-II B-I MQ-I SUP-II a b c d

Total protein (mg)

Specific activity (BAEE units/mg) a

Total activity (BAEE units × 103) b

Recovery percentage (%) c

Purification fold d

5800 2562 607 65 33

255 402 1120 6380 8520

1479.0 1029.9 679.8 414.7 281.2

100 69.6 46.0 28.0 19

1.0 1.6 4.4 25.0 33.4

Specific activity (trypsin inhibitory activity) of KBTI was tested by using BAEE as substrate. Total activity = specific activity × total protein of the fraction. Recovery percentage = (total activity of the fraction / total activity of crude extract) × 100%. Purification fold = specific activity of the fraction / specific activity of crude extract.

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TABLE 3. Comparison of the N-terminal amino acid sequences of KBTI and other Kunitz type trypsin inhibitors mostly from the same (Fabaceae) family. No.

Name

1 KBTI 2 SBTI-1 3 SBTI-2 4 PDTI-1 5 PDTI-2 6 PATI 7 DMTI-1 8 DMTI-2 9 PJTI 10 WTI-1 11 PFTI 12 PRTI 13 BTI-1 14 BTI-2 15 ESTI Concordant residue Concordance (%)

N-terminal amino acid sequence 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

D D D D D D

P V V V V V V Y L L L T Q Q E V

L L L L L L F D L V K L S S V L

D D D D D D D S D D D P C C L D

N N N A A A T D V V M W G G D N/A

E E E E E E E G D E E K K K S E

H G G G G G G F G G G V Q Q D G

N N N K I K N P E K D E E E G N/E

M P P F F F G L I T I G W W H F

L L L L L L I R L V L E P P M L

E E S L R X R N R R E S E E L E

N N N N N N N G N N N Q L L R N

G G G G G G G G G G G E V V N G

G G G G G E G

D

F F F F F F Q V E E E S R R E F

50

40

40

53

67

20

60

60

27

20

53

33

67

73

80

L

F Y L L

G G G V G G G G

Identity (%) – 80 73 60 60 53 40 6.7 40 33 47 6.7 13 13 6.7

NOTES: SBTI-1 (40) and SBTI-2 (41) are trypsin inhibitors (TI) from soybean seeds; PDTI-1 (42) and PDTI-2 (43) are purified from Peltophorum dubium; PATI: from Peltophorum africanum (weeping wattle) seeds (44); DMTI-1 and DMTI-II: from Dimorphandra mollis (Leguminosae-Mimosoideae) (45, 46); PJTI: Prosopsis juliflora trypsin inhibitor (47); WTI-1: trypsin inhibitors (WTI-1A and WTI-1B) from winged bean seeds (Psophocarpus tetragonolobus (L)DC.) (48); PFTI: TI from Plathymenia foliolosa seeds (16); PRTI: TI-peptide2 from Putranjiva roxburghii seeds (49); BTI-1 and BTI-2: TIs from seeds of buckwheat (Fagopyrum esculentum Monch) (50); ESTI: TI from sword bean (Entada scandens) (51).

positive control of this assay manifested an IC50 value of 2.2 μM which compared favorably with the previously reported results (26). Furthermore, KBTI induced a dose-dependent cytokine expression in mouse splenocytes (Fig. 6). Results showed that KBTI significantly stimulated TNF-α, IL-1β and INF-γ expression at the mRNA level at a final concentration of 20 μg/ml, when the concentration was raised to 100 μg/ml, a band of IL-2 could be seen and its INF-γ inducing activity was more apparent. KBTI weakly inhibited the proliferation of CNE-2, HNE-2, MCF-7 and HepG2 cells in a dose-dependent manner (Figs. 7A–D).

FIG. 3. Thermal and pH stability tests of KBTI. (A) pH stability of KBTI was measured after incubation at different pH values for 0.5 h at 37 °C. (B) Temperature stability was tested after incubation for 30 min from 0 to 100 °C. Results represent mean ± S.D. (n = 3).

DISCUSSION Kunitz type inhibitors (bovine pancreatic and soybean trypsin inhibitors), Bowman–Birk type inhibitors, potato type I and II inhibitors, and Kazal type inhibitors (pancreatic secretory trypsin inhibitors) constitute the classical serine protease inhibitor family (11). Among them, Kunitz type trypsin inhibitors are a class of small (Mr ∼ 20 kDa) water-soluble proteins with a low cysteine content and a single reactive site (31). They have been reported with efficacy against tumor cells in vitro, in animal models, and in human phase IIa clinical trials (11). In this study, a 20-kDa Kunitz trypsin inhibitor KBTI was purified from Korean black soybeans. KBTI is pH- and thermostable and its trypsin inhibitory activity can be decreased by DTT in accordance with previous reports, signifying the importance of disulfide bonds to trypsin inhibitory activity (11, 16). Like broad bean trypsin inhibitor (32), KBTI is capable of inhibiting chymotrypsin. The N-terminal sequence of KBTI is highly homologous to those of related Kunitz type trypsin inhibitors. The first 15 N-terminal amino acids disclosed some conserved sequences [DF-LDN(A)E-N(E)LENGG] which is in accordance with some reported Kunitz type trypsin inhibitors. HIV-reverse transcriptase (RT) is essential for HIV replication. It catalyzes the conversion of RNA of the reverse-transcribing RNA virus into DNA, which is then incorporated into the host genome. RT is recognized as a useful molecular target for HIV therapy (33-35). KBTI inhibits the activity of HIV-1-RT with an IC50 of 0.71 μM (Fig. 5). The IC50 value is smaller than some of the previously purified anti-HIV-1RT proteins, such as a 8-kDa Bowman–Birk trypsin inhibitor from Hokkaido large black soybeans (IC50 value is 38 μM) (14) and the 62-kDa pinto bean lectin (IC50 value is 2.2 μM) (26). It seems that KBTI may have some potential to be developed into a therapeutic agent for AIDS. Cytokines are a category of signaling molecules and mediate a number of functions, ranging from effects on cell growth, differentiation, survival, and a number of effector activities (36). Proteins with immunostimulatory functions such as activation of monocytes/macrophages, cytokine release and induction of apoptosis are believed to be beneficial for defense against tumors (37). KBTI at 20 μg/ml (1 μM) and 100 μg/ml (5 μM) (low and non-toxic levels tested by MTT test, data not shown) could dose-dependently induce

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FIG. 6. Induction of cytokine mRNA expression by KBTI. Mouse splenocytes were treated with KBTI at final concentrations of 100 or 20 μg/ml for 4 h. Its cytokineinducing activity was measured by RT-PCR. The experiment was performed in triplicate and one set of typical results is shown.

FIG. 4. Trypsin and chymotrypsin inhibitory activities of KBTI and the effect of dithiothreitol (DTT) treatment. After incubation with KBTI at different concentrations for 30 min, the remaining activity of (A) bovine pancreatic trypsin or (B) αchymotrypsin was measured using casein as substrate. Its kinetic stability on exposure to DTT was also assayed (C). soybean trypsin inhibitor from USB Corporation (TI) was used as positive control. The asterisk denotes p b 0.05 compared with control. Results represent mean ± SD (n = 3).

the mRNA expression of pro-inflammatory cytokines including TNF-α, IL-1β, IL-2 and INF-γ. Our further in vitro studies disclosed the antiproliferative activity of KBTI against some tumor cell lines including NPC cell lines. Nasopharyngeal carcinoma is rare in most parts of the world but it has a high incidence in South China and Southeast Asia (38), and during 2000–2004, this cancer reaches an average age-standardized incidence rate of 11.8 per 100,000 person-5 years in Hong Kong (39). The results of the MTT assay show that KBTI weakly inhibited proliferation of NPC CNE-2 and HNE-2 cells. Besides, it also showed slight antiproliferative activity on MCF-7 breast cells and Hep G2 hepatoma cells. In contrast to the antifungal Bowman–Birk trypsin inhibitor from broad beans (32), KBTI is devoid of antifungal activity. Other leguminous trypsin inhibitors also do not inhibit fungal growth (14, 15). In summary, by using the combined anion exchange, Affi-gel Blue gel and gel filtration chromatography, a 20-kDa trypsin inhibitor (KBTI) was purified from a new cultivar of soybean, Korean large black soybean. It was pH and heat stable and exhibited novel characteristics of the Kunitz type trypsin inhibitors. Besides HIV-1-RT inhibitory activity, the KBTI manifested immunostimulatory activity and inhibited growth of tumor cell. ACKNOWLEDGMENTS We thank Eric C.H. Yau and Janis Ching for their secretarial assistance. References

FIG. 5. HIV-1 reverse transcriptase inhibitory activity of KBTI. Pinto bean lectin was used as positive control. Results represent mean ± SD (n = 3).

1. Di Giacomo, C., Acquaviva, R., Sorrenti, V., Vanella, A., Grasso, S., Barcellona, M. L., Galvano, F., Vanella, L., and Renis, M.: Oxidative and antioxidant status in plasma of runners: effect of oral supplementation with natural antioxidants, J. Med. Food, 12, 145–150 (2009). 2. Hsieh, H. M., Wu, W. M., and Hu, M. L.: Soy isoflavones attenuate oxidative stress and improve parameters related to aging and Alzheimer's disease in C57BL/6J mice treated with D-galactose, Food. Chem. Toxicol., 47, 625–632 (2009). 3. Retelny, V. S., Neuendorf, A., and Roth, J. L.: Nutrition protocols for the prevention of cardiovascular disease, Nutr. Clin. Pract., 23, 468–476 (2008). 4. Martin, D., Song, J., Mark, C., and Eyster, K.: Understanding the cardiovascular actions of soy isoflavones: potential novel targets for antihypertensive drug development, Cardiovasc. Hematol. Disord. Drug Targets, 8, 297–312 (2008). 5. Orgaard, A. and Jensen, L.: The effects of soy isoflavones on obesity, Exp. Biol. Med. (Maywood), 233, 1066–1080 (2008).

216

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FIG. 7. Antiproliferative activity of KBTI on (A) CNE-2 and (B) HNE-2 nasopharyngeal carcinoma cells, (C) MCF-7 breast cancer cells and (D) Hep G2 hepatoma cells. The antiproliferative activity of KBL was measured by MTT assay and the dose– and time–response relationships are shown. Results represent mean ± SD (n = 3).

6. Jang, E. H., Moon, J. S., Ko, J. H., Ahn, C. W., Lee, H. H., Shin, J. K., Park, C. S., and Kang, J. H.: Novel black soy peptides with antiobesity effects: activation of leptinlike signaling and AMP-activated protein kinase, Int. J. Obes. (Lond), 32, 1161–1170 (2008). 7. Ko, K. P., Park, S. K., Cho, L. Y., Gwack, J., Yang, J. J., Shin, A., Kim, C. S., Kim, Y., Kang, D., Chang, S. H., Shin, H. R., and Yoo, K. Y.: Soybean product intake modifies the association between interleukin-10 genetic polymorphisms and gastric cancer risk, J. Nutr., 139, 1008–1012 (2009). 8. Murooka, Y. and Yamshita, M.: Traditional healthful fermented products of Japan, J. Ind. Microbiol. Biotechnol., 35, 791–798 (2008). 9. Ravindranath, M. H., Muthugounder, S., Presser, N., and Viswanathan, S.: Anticancer therapeutic potential of soy isoflavone, genistein, Adv. Exp. Med. Biol., 546, 121–165 (2004). 10. Low Dog, T.: Menopause: a review of botanical dietary supplements, Am. J. Med., 118, (Suppl. 12B) 98–108 (2005). 11. Losso, J. N.: The biochemical and functional food properties of the Bowman–Birk inhibitor, Crit. Rev. Food Sci. Nutr., 48, 94–118 (2008). 12. Birk, Y.: Protein proteinase inhibitors in legume seeds—overview, Arch. Latinoam. Nutr., 44, 26S–30S (1996). 13. Fritz, H.: Foreword, in: K. von der Helm, B. D. Korant, J. C. Cheronis (Eds.), Proteases as Targets for Therapy, Springer-Verlag, Berlin, 2000, pp. 1–6 (2000), 2000. 14. Ho, V. S. and Ng, T. B.: A Bowman–Birk trypsin inhibitor with antiproliferative activity from Hokkaido large black soybeans, J. Pept. Sci., 14, 278–282 (2008). 15. Cheung, A. H., Wong, J. H., and Ng, T. B.: Trypsin-chymotrypsin inhibitors from Vigna mungo seeds, Protein Pept. Lett., 16, 277–284 (2009). 16. Ramos Vda, S., Silva Gde, S., Freire, M. G., Machado, O. L., Parra, J. R., and Macedo, M. L.: Purification and characterization of a trypsin inhibitor from Plathymenia foliolosa seeds, J. Agric. Food Chem., 56, 11348–11355 (2008). 17. Wang, H. X. and Ng, T. B.: Concurrent isolation of a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots, Biochem. Biophys. Res. Commun., 342, 349–353 (2006). 18. Nielsen, T. B. and Reynolds, J. A.: Measurements of molecular weights by gel electrophoresis, Methods Enzymol., 48, 3–10 (1978). 19. Schelud'ko, A. V., Makrushin, K. V., Tugarova, A. V., Krestinenko, V. A., Panasenko, V. I., Antonyuk, L. P., and Katsy, E. I.: Changes in motility of the rhizobacterium Azospirillum brasilense in the presence of plant lectins, Microbiol. Res., 164, 149–156 (2009). 20. Lin, P. and Ng, T. B.: Preparation and biological properties of a melibiose binding lectin from Bauhinia variegata seeds, J. Agric. Food. Chem., 56, 10481–10486 (2008). 21. Lam, S. S., Wang, H., and Ng, T. B.: Purification and characterization of novel ribosome inactivating proteins, alpha- and beta-pisavins, from seeds of the garden pea Pisum sativum, Biochem. Biophys. Res. Commun., 253, 135–142 (1998). 22. Bergmeyer, H. U., Gawehn, K., and Grassl, M.: Methods of enzymatic analysis, 2 ed.Academic Press, Inc., New York, NY, 1974.

23. Ng, T. B. and Ngai, P. H.: The trypsin-inhibitory, immunostimulatory and antiproliferative activities of a napin-like polypeptide from Chinese cabbage seeds, J. Pept. Sci., 10, 103–108 (2004). 24. Shih, C. Y., Khan, A. A., Jia, S., Wu, J., and Shih, D. S.: Purification, characterization, and molecular cloning of a chitinase from the seeds of Benincasa hispida, Biosci. Biotechnol. Biochem., 65, 501–509 (2001). 25. Collins, R. A., Ng, T. B., Fong, W. P., Wan, C. C., and Yeung, H. W.: A comparison of human immunodeficiency virus type 1 inhibition by partially purified aqueous extracts of Chinese medicinal herbs, Life Sci., 60, PL345–PL351 (1997). 26. Wong, J. H., Wong, C. C., and Ng, T. B.: Purification and characterization of a galactose-specific lectin with mitogenic activity from pinto beans, Biochim. Biophys. Acta, 1760, 808–813 (2006). 27. Cheung, A. H., Wong, J. H., and Ng, T. B.: Musa acuminata (Del Monte banana) lectin is a fructose-binding lectin with cytokine-inducing activity, Phytomedicine, 16, 594–600 (2009). 28. Zhang, S., Lin, Z. N., Yang, C. F., Shi, X., Ong, C. N., and Shen, H. M.: Suppressed NF-kappaB and sustained JNK activation contribute to the sensitization effect of parthenolide to TNF-alpha-induced apoptosis in human cancer cells, Carcinogenesis, 25, 2191–2199 (2004). 29. Lin, P. and Ng, T. B.: A novel and exploitable antifungal peptide from kale (Brassica alboglabra) seeds, Peptides, 29, 1664–1671 (2008). 30. Wang, H. X. and Ng, T. B.: A ribonuclease from Chinese ginseng (Panax ginseng) flowers, Protein Expr. Purif., 33, 195–199 (2004). 31. Laskowski, M. and Qasim, M. A.: What can the structures of enzyme-inhibitor complexes tell us about the structures of enzyme substrate complexes? Biochim. Biophys. Acta, 1477, 324–337 (2000). 32. Ye, X. Y. and Ng, T. B.: A new peptidic protease inhibitor from Vicia faba seeds exhibits antifungal, HIV-1 reverse transcriptase inhibiting and mitogenic activities, J. Pept. Sci., 8, 656–662 (2002). 33. Barre-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W., and Montagnier, L.: Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS), Science, 220, 868–871 (1983). 34. Kati, W. M., Johnson, K. A., Jerva, L. F., and Anderson, K. S.: Mechanism and fidelity of HIV reverse transcriptase, J. Biol. Chem., 267, 25988–25997 (1992). 35. Karpas, A.: Human retroviruses in leukaemia and AIDS: reflections on their discovery, biology and epidemiology, Biol. Rev. Camb. Philos. Soc., 79, 911–933 (2004). 36. Taub, D. D.: Cytokine, growth factor, and chemokine ligand database, Curr. Protoc. Immunol., 29, Chapter 6, Unit 6. 37. Lyu, S. Y. and Park, W. B.: Effects of Korean mistletoe lectin (Viscum album coloratum) on proliferation and cytokine expression in human peripheral blood mononuclear cells and T-lymphocytes, Arch. Pharm. Res., 30, 1252–1264 (2007).

VOL. 109, 2010 38. Ma, B. B., Hui, E. P., and Chan, A. T.: Systemic approach to improving treatment outcome in nasopharyngeal carcinoma: current and future directions, Cancer Sci., 99, 1311–1318 (2008). 39. Law, C. K. and Mang, O.: Cancer incidence in Hong Kong, Med. Bull., 12, 18–21 (2007). 40. Koide, T. and Ikenaka, T.: Studies on soybean trypsin inhibitors: 3. Amino-acid sequences of the carboxyl-terminal region and the complete amino-acid sequence of soybean trypsin inhibitor (Kunitz), Eur. J. Biochem., 32, 417–431 (1973). 41. Kim, S. H., Hara, S., Hase, S., Ikenaka, T., Toda, H., Kitamura, K., and Kaizuma, N.: Comparative study on amino acid sequences of Kunitz-type soybean trypsin inhibitors, Tia, Tib, and Tic, J. Biochem., 98, 435–448 (1985). 42. Rodrigues Macedo, M. L., Machado Freire, M. G., Cabrini, E. C., Toyama, M. H., Novello, J. C., and Marangoni, S.: A trypsin inhibitor from Peltophorum dubium seeds active against pest proteases and its effect on the survival of Anagasta kuehniella (Lepidoptera: Pyralidae), Biochim. Biophys. Acta, 1621, 170–182 (2003). 43. Fernanda Troncoso, M., Cerda Zolezzi, P., Hellman, U., and Wolfenstein-Todel, C.: A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis, Arch. Biochem. Biophys., 411, 93–104 (2003). 44. Joubert, F. J.: Purification and some properties of a proteinase inhibitor (DE-1) from Peltophorum africanum (weeping wattle) seed, Hoppe. Seylers. Z. Physiol. Chem., 362, 1515–1521 (1981).

TRYSIN INHIBITOR FOR KOREAN SOYBEANS

217

45. Macedo, M. L., de Matos, D. G., Machado, O. L., Marangoni, S., and Novello, J. C.: Trypsin inhibitor from Dimorphandra mollis seeds: purification and properties, Phytochemistry, 54, 553–558 (2000). 46. Mello, G. C., Oliva, M. L., Sumikawa, J. T., Machado, O. L., Marangoni, S., Novello, J. C., and Macedo, M. L.: Purification and characterization of a new trypsin inhibitor from Dimorphandra mollis seeds, J. Protein Chem., 20, 625–632 (2001). 47. Negreiros, A. N., Carvalho, M. M., Xavier Filho, J., Blanco-Labra, A., Shewry, P. R., and Richardson, M.: The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora, Phytochemistry, 30, 2829–2833 (1991). 48. Yamamoto, M., Hara, S., and Ikenaka, T.: Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L)DC.), J. Biochem., 94, 849–863 (1983). 49. Chaudhary, N. S., Shee, C., Islam, A., Ahmad, F., Yernool, D., Kumar, P., and Sharma, A. K.: Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds, Phytochemistry, 69, 2120–2126 (2008). 50. Pandya, M. J., Smith, D. A., Yarwood, A., Gilroy, J., and Richardson, M.: Complete amino acid sequences of two trypsin inhibitors from buckwheat seed, Phytochemistry, 43, 327–331 (1996). 51. Lingaraju, M. H. and Gowda, L. R.: A Kunitz trypsin inhibitor of Entada scandens seeds: another member with single disulfide bridge, Biochim. Biophys. Acta, 1784, 850–855 (2008).