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Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications
Biochemical and Biophysical Research Communications 396 (2010) 806–811
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Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications Evandro Fei Fang a, Jack Ho Wong a, Clara Shui Fern Bah b, Peng Lin a, Sai Wah Tsao c, Tzi Bun Ng a,* a
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China Department of Food Science, Division of Sciences, University of Otago, New Zealand c Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Sassoon Road, Pokfulam, Hong Kong SAR, China b
a r t i c l e
i n f o
Article history: Received 13 April 2010 Available online 8 May 2010 Keywords: Bauhinia variegata var. variegata Proteinase inhibitors Cytokines HIV Nasopharyngeal carcinoma
a b s t r a c t Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel’s foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (Ki, 0.1 109 M) of BvvTI ranks the highest among them. Besides anti-HIV1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Bauhinia variegata (Camel’s foot tree), a species of flowering plant belonging to the family Leguminosae, has long been cultivated as an ornamental tree in Hong Kong [1]. It is one of the three common Bauhinia tree species in Hong Kong, the other two being Bauhinia purpurea (Purple camel’s foot) and Bauhinia blakeana Dunn (the Hong Kong Orchid Tree) [2]. Studies on the varieties of B. variegata have indicated that a number of medicinal components can be purified from the plant. Its stem displays antibacterial, antifungal, as well as anti-tumor activities while its roots have antiinflammatory activity [3,4]. As legume seeds usually have a high content of serine protease inhibitors, it is therefore not surprising that serine protease inhibitors have been identified in different varieties of B. variegata [5–7]. Serine protease inhibitors (serpins) from leguminous seeds inactivate several enzymes, and are classified into families including Kunitz trypsin inhibitors (TIs), Bowman–Birk TI and others, depending on their molecular mass and cysteine content [8,9]. Kunitz-type TIs are characterized by their ability to inhibit proteases that are involved in physiological events such as coagulation, inflammation, and cancer [9]. Due to the involvement of proteases in several pathological events, characterizing the structure and function of new protease inhibitors may identify important tools for the fields of disease mechanism exploration to drug design [10,11]. * Corresponding author. Address: Room 302B, BMSB, The Chinese University of Hong Kong, Hong Kong, China. Fax: +852 26035123. E-mail addresses: [email protected], [email protected] (T.B. Ng). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.140
Here, we for the first time describe the isolation, biochemical and functional properties of a TI, BvvTI, from the seeds of a local variety of B. variegata (var. variegata). The results show BvvTI has significant anti-HIV-1 reverse transcriptase and anti-nasopharyngeal carcinoma CNE-1 properties and unveils its potential medicinal applications. 2. Materials and methods 2.1. Protein purification Mature seeds (250 g) of B. variegata var. variegata were harvested on the campus of the Chinese University of Hong Kong (CUHK) and authenticated by Prof. Shiu Ying Hu, honorary Professor of Chinese Medicine, CUHK. The supernatant derived from centrifugation of the seed homogenate was first loaded onto a SPSepharose column (GE Healthcare, UK). The adsorbed faction was then loaded on a Mono S 5/50 GL column, followed on a Superdex 75 10/300 GL column, using an AKTA Explorer 100 chromatography system (GE Healthcare, UK). The purified BvvTI in peak BVSUPI was dialyzed extensively, freeze-dried, and stored at 20 °C for further studies. 2.2. Molecular mass and N-terminal amino acid sequence determination Both sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for molecular
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weight determination were carried out according to methods described in Ref. [12]. N-terminal amino acid sequence analysis was performed using a HP 1000A Edman degradation unit and a HP 1000 HPLC system [12]. The percentage of sequence identity with TIs from related legumes was determined using BCM search launcher, the software ClustalX 1.83, and the on line BOXSHADE 3.21 server [13].
against trypsin was tested and compared with a negative control. Reagents used included phenylglyoxal (PG, for arginine resides modification) [20], phenylmethylsulfonyl fluoride (PMSF, for serine modification), 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB, for reduction of the thiol groups), sodium borohydride (SB, for lysine modification), and N-acetylimidazole (NA, for tyrosine residues modification) [19].
2.3. Enzymatic analysis
2.6. Assay of HIV-1 reverse transcriptase (HIV-1-RT) inhibitory activity
2.3.1. With specific synthetic substrate The assay was done by using a spectrophotometric method with some modifications [12,14,15]. Firstly, different doses of 20 lL BvvTI in 50 mM Tris–HCl (pH 7.6, final concentrations from 0.125, 0.25, 0.5, 0.75, to 1 nM) were incubated with 40 lL 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 of each tube was measured by adding 1.44 mL N-a-benzoyl-L-arginine ethyl ester (BAEE, two different final concentrations from 0.5 to 1 mM) as substrate. After immediately mixing by inversion, the increase in A253 was recorded for 5 min (37 °C). Reaction without the addition of sample was used as a control:
The assay was performed using an HIV-1-RT (recombinant) ELISA kit, following the instructions of the manufacturer (Boehringer Mannheim, Germany) [12]. Pinto bean lectin with anti-HIV-1-RT activity was chosen as a positive control [21].
Calculation : BAEE units=mg trypsin inhibitor ¼ ðDA253=min Positive DA253=min Sample TestedÞ=½ð0:001Þð0:04Þ ðsampleÞ where (0.001) = the change in A253/min per unit of trypsin at pH 7.6 and 37 °C in a 1.5 mL reaction mixture; (0.04) or (sample) = solid (mg) of trypsin or sample used in the reaction. In addition, 1 U 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 37 °C. Chymotrypsin inhibitory activity was determined using the same assay, but by adding more BvvTI (final concentrations from 12.5, 25, 50, 75, to 100 nM) and using N-benzoyl-L-tyrosine ethyl ester (BTEE) as the substrate instead. One a-chymotrypsin unit hydrolyzes 1.0 lmol of BTEE per minute at pH 7.6 and 37 °C. Inhibition constants (Ki) of BvvTI against both trypsin and achymotrypsin were determined as described elsewhere [16]. The Ki value was estimated by using Sigma Plot 10.0, Enzyme Kinetics Module 1.3 (Systat Software Inc., San Jose, USA). 2.3.2. With casein as the substrate In addition, 1% casein was used as substrate for the description of a dose-dependent relationship between BvvTI and residual trypsin activity as mentioned previously [15,17]. Soybean trypsin inhibitor from USB Corporation was used as a positive control.
2.7. Assay of antiproliferative and apoptosis-inducing activity All seven cell lines including six tumor cell lines (human hepatocellular carcinoma Hep G2, human breast tumor MCF-7, mouse leukemia L1210, and three human nasopharyngeal carcinoma CNE-1, CNE-2, and HNE-2 cell lines) and one transformed normal human nasopharyngeal epithelial cell line NP 69 were cultured as previously described [12]. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test was used to monitor inhibition of cell growth following the method reported elsewhere [22]. Apoptosis was observed by chromatin staining with Hoechst 33342 (Sigma, USA) by using the cell line CNE-1 as a paradigm. 2.8. Assay of cytokine-inducing activity Splenocytes from male BALB/c mice were prepared for the assay. After extraction of total RNA using TRIZOLÒ (Invitrogen, USA), changes of cytokine expression at the mRNA level was measured by using a reverse transcriptase PCR kit (GeneAmpÒ, USA). Cytokines including tumor necrosis factor alpha (TNF-a), interleukin-1b (IL-1b), interleukin-2 (IL-2), and interferon-gamma (INF-c) were measured [23]. 2.9. Statistical analysis Results were taken from three independent experiments performed in triplicate, and data were expressed as means ± standard deviation (SD). The median inhibition concentration (IC50) was determined using SPSS 11.0.1 statistical software (SPSS Inc., Chicago, USA). For between-group comparisons, Student’s t-test or one-way ANOVA was used where appropriate and differences with p values of 0.05 or less were considered to be statistically significant. 3. Results
2.4. Thermal and pH stability 3.1. Isolation of BvvTI Thermal and pH stability assays were conducted as previously reported [12,18]. Remaining trypsin inhibitory activity was calculated as percent of the value at 30 °C or pH 7. 2.5. Reactive sites determination The effect of the reducing agent dithiothreitol (DTT) on the stability of BvvTI was examined following the method of Ramos et al. [18]. Soybean TI was used as a positive control. Chemical modification of amino acid residues on the trypsin inhibitory activity of BvvTI was carried out as previously described [16,19]. After treatment with different reagents, the residual inhibitory activity
BvvTI was purified using the method shown in Fig. 1A and Supplementary Fig. 1. The crude seed extract was first run on a SP-Sepharose column, followed by FPLC on a Mono S column. Five major adsorbed peaks (Supplementary Fig. 1A) were obtained. The inhibitor-containing peak, BV-MSIV, was subsequently subjected to size exclusion chromatography on Superdex 75, and purified BvvTI was acquired from peak BV-SUPI. The purity of the BvvTI was confirmed by presence of a sharp single peak in both FPLC-gel filtration on Superdex 75 (data not shown) and mass spectrometry (Fig. 1C), and a single band in SDS–PAGE (Fig. 1B lane 4). Data in Table 1 and Fig. 1 tracked the purification steps of BvvTI.
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Fig. 1. Purification of BvvTI. (A) Schematic representation of the chromatographic steps used to purify BvvTV. (B) SDS–PAGE showing purity and molecular weight of BvvTI. Lane 1, B. variegata var. variegata crude extract (20 lg); lane 2, fraction adsorbed on SP-Sepharose (30 lg); lane 3, peak BV-MSIV from FPLC-Mono-S column (20 lg); lane 4, peak BV-SUPI from Superdex 75 column (20 lg); and lane 5, molecular weight marker from GE Healthcare. (C) The MALDI-TOF mass spectrometry results of BvvTI showing a peak of 21,096 Da.
Table 1 Summary of purification of BvvTI from 250 g mature seeds of Bauhinia variegata var. variegata. Sample
Step (peak)
Total protein (mg)
Specific trypsin inhibitory activity (BAEE units/mg)
Purification fold*
Yield (%)
BvvTI
Water extract Sp-Sepharose Mono S (BV-MSIV) Superdex 75 (BV-SUPI)
10,200 3824 692 429
294 890 3320 5865
1.0 3.0 11.3 20.0
100 37.5 6.8 4.2
*Purification fold equals specific trypsin inhibitory activity of the chromatographic fraction divided by specific trypsin inhibitory activity of the water extract fraction.
3.2. Biological and enzymatic activities of BvvTI
3.4. Anti-HIV-1-RT activity
SDS–PAGE of acquired BvvTI displayed only a single band which revealed that the protein was homogeneous and was a single 21-kDa polypeptide chain in accordance with the MALDI-TOF MS results (Fig. 1B and C). The sequence of the first 20 N-terminal amino acids of BvvTI is as follows: DTLLD TDGEV VRNSG GRYYI. As shown in Supplementary Table 1, in comparison with Kunitz TIs from other Bauhinia species, a pronounced homology was observed. By using specific synthetic substrates, the inhibitory activities of BvvTI towards trypsin and a-chymotrypsin were 5865 BAEE units/ mg and 43 BTEE units/mg, respectively (Table 1). In addition, a relationship between BvvTI dose and residual trypsin or a-chymotrypsin activity is shown in Supplementary Fig. 2 when casein was used as a substrate. Lineweaver–Burk double reciprocal plots (Supplementary Fig. 3A and C) and Dixon plots (Supplementary Fig. 3B and D) showed a non-competitive-type inhibitory activity of BvvTI against both trypsin and a-chymotrypsin and with Kis of 0.1 109 M and 250.5 109 M, respectively.
In vitro ELISA results revealed that BvvTI could inhibit the activity of HIV-1-RT with an IC50 value of 6.4 lM (Supplementary Fig. 4). Results of the positive control, the pinto bean lectin, yielded an IC50 value of 2.5 lM which compares well with previously reported values [21].
3.3. Stability of BvvTI and its potential reactive site residues Fig. 2A, B and C show the thermal, pH and chemical stabilities of BvvTI. The trypsin inhibitory activity of BvvTI was stable up to 50 °C and in buffers with pH 4–12. Furthermore, BvvTI was able to withstand reducing conditions of 1 mM DTT for 0.5 h, but there was a steady loss of activity to 89% and 27% of the initial activity when the DTT concentration was augmented to 10 mM and 100 mM, respectively (Fig. 2C). As shown in Fig. 2D, modifications of both arginine residues and serine residues brought about serious reduction (about 75% and 55%, respectively) of its trypsin inhibitory activities. However, no significant loss of activity was observed after modification of thiol groups, lysine or tyrosine residues.
3.5. Anti-tumor and cytokine-inducing activities As listed in Table 2, the MTT assay revealed that BvvTI exerted a specific antiproliferative activity on CNE-1 cells in a time-dependent manner. There was no significant effect on other tumor cells and the normal cell line NP 69 even after 48 h of exposure. CNE1 was further chosen to study the apoptosis-inducing ability of BvvTI. Fig. 3 shows the morphological changes of CNE-1 cells when the BvvTI concentration was increased from 0 to 100 lM, and the reduction of cell numbers and apoptotic bodies were noticed accordingly (Fig. 3). BvvTI differentially regulates the mRNA expression of cytokines including IL-1b, IL-2, TNF-a, and INF-c. As shown in Fig. 4, BvvTI significantly increased the transcriptional levels of IL-1b, IL-2 and TNF-a in a dose-dependent effect at a specified time point. However, the cytokine-inducing ability towards INF-c was not significant. 4. Discussion BvvTI was isolated by employing a simple liquid chromatography method with an acceptable harvest rate of approximately 5% (Table 1) similar to that achieved using other methods [6]. The biological characteristics of BvvTI demonstrate similarity with other Bauhinia Kunitz-type TIs, including an identical molecular weight (Fig. 1) and a high degree of homology of N-terminal amino acid sequence (Supplementary Table 1) [6,7,24].
809
A 120
B 120
Residual trypsin inhibitory activity (%)
Residual trypsin inhibitory activity (%)
E.F. Fang et al. / Biochemical and Biophysical Research Communications 396 (2010) 806–811
100
* *
80
60
*
40
20
* *
0
100
* 80
*
60
20 0
0
20
40
60
80
100
120
0
2
4
6
Temperature
D BvvTI
Soybean TI
* *
80
60
*
40
*
20
0
1
0
10
100
DTT (mM)
Residual trypsin inhibitory activity (%)
Residual trypsin inhibitory activity (%)
C 120 100
8
10
12
14
pH
120
100
80
*
60
40
*
20
0
B B G TI SF NA TN +S Bvv TI + P + PM TI + + D vvTI v v I I v v T T B B B Bv v Bvv
Fig. 2. Stability of BvvTI and its potential reactive site residues. (A) Thermal stability was tested after BvvTI (50 lM in Tris–HCl buffer) had been exposed to 0–100 °C for 30 min. (B) pH stability of BvvTI was measured after dissolving the lyophilized protein in buffers of different values to a final concentration of 50 lM, followed by incubation for 0.5 h at 37 °C. (C) BvvTI were incubated with dithiothreitol (DTT) at different final concentrations (0, 1, 10, and 100 mM) for 30 min at 37 °C. Soybean TI was used as a positive control. (D) Chemical modification of amino acid residues on the trypsin inhibitory activity of BvvTI. Reagents in details were in Section 2.5. Residual trypsin inhibitory activity in all (A), (B) (C) and (D) was measured by using BAEE as substrate. Means ± SD of three independent experiments are shown. *p < 0.05 versus the residual activity at 30 °C (A) or pH 7 (B) or negative control (C, D), respectively.
Table 2 Antiproliferative activity of BvvTI on different tumor cell lines. Tumor cell line
Hep G2 MCF-7 L1210 CNE-1 CNE-2 HNE-2 NP 69 (normal)
IC50 (lM) 24 h
48 h
225.36 ± 27.44 – – 54.27 ± 4.29 – – –
87.89 ± 5.64* – – 21.37 ± 2.36* 354.70 ± 24.89* – –
Values of IC50 represent means ± SD of three determinations using the MTT assay. –, antiproliferative activity was undetectable at BvvTI concentrations above 1 mM. * p < 0.05 compared with IC50 (24 h) of BvvTI for the same cell line.
Results obtained by using specific synthetic substrates (BAEE and BTEE) and the non-specific substrate casein, manifest a significant trypsin inhibitory activity and a slighter antiproteolytic activity against chymotrypsin (Table 1, and Supplementary Figs. 2 and 3). This was to some extent similar to other Kunitz TIs [6,7,18,24– 26]. To the best of our knowledge, the Ki (0.1 109 M) of BvvTI to-
wards trypsin is so far the highest among Bauhinia serine protease inhibitors [5–7,9,24]. For example, the Ki values towards trypsin for BvcTI (B. variegata var. candida TI) and BvlTI (B. variegata var. lilac TI) are 6.9 and 1.2 nM, respectively [6]. A much bigger Ki value observed for a-chymotrypsin, unveiled a higher affinity between BvvTI and trypsin, in accordance with previous literature on Bauhinia [6,7,9,24]. Several structural features are conserved in most Kunitz-type inhibitors, including molecular mass (20 kDa), four cysteine residues arranged in two disulfide bridges, and the single reactive site (Arg-Ser or Arg-Ile) [5,6]. However, there are some exceptions. For instance, two 18-kDa protease inhibitors from Bauhinia bauhinioides are devoid of disulfide bridges and with different reactive site [5]. For BvvTI, DTT treatment (break down the disulfide bonds) resulted in a gradual loss of trypsin inhibitory activity, which sheds light on the importance of the disulfide bond to its trypsin/a-chymotrypsin inhibitory activities [18]. Furthermore, chemical modification studies were carried out to identify the amino acids responsible for the inhibitory activity of BvvTI against trypsin. The results (Fig. 2D) manifest that both Arg and Ser residues present in the reactive site play a significant role in its antiproteolytic activity towards trypsin, which was coincidence with the reactive
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Fig. 3. Dose-escalation study of BvvTI-induced nuclear morphological changes in CNE-1 cells. After treatment with different doses of BvvTI including (A) 0 lM (control), (B) 10 lM, (C) 50 lM, and (D) 100 lM for 24 h, CNE-1 cells were stained with 1 lg/mL Hoechst 33342 for 10 min and the morphological changes of cells were examined by fluorescence microscopy. Row 1, brightfield images; row 2, fluorescent images; row 3, merged images between row 1 and row 2; row 4, 5-fold zoom of merged images (row 3) as indicated by thin arrows. Typical apoptotic bodies in D-4 are indicated by arrows. Bar in merged image indicates 20 lm.
Fig. 4. Induction of cytokine mRNA expression by BvvTI. Mouse splenocytes were treated with BvvTI at final concentrations of 4 or 20 lM for 4–20 h. Its cytokine-inducing activity was measured by RT-PCR. The experiment was performed in triplicate and one set of typical results is shown (A). Lane 1, DNA ladder; lane 2, control (splenocytes incubated for 16 h without any treatment); lane 3, splenocytes treated with 4 lM BvvTI for 4 h; lane 4, 20 lM BvvTI for 4 h; lane 5, 4 lM BvvTI for16 h; and lane 6, 20 lM BvvTI for 16 h. Fig. (B) RT-PCR results from three independent experiments. Band intensity was measured using Alphaimager 2200 in which each cytokine mRNA expression was normalized relative to the GAPHD expression levels. *p < 0.05 compared with control, p < 0.05 treatment with 20 lM compared with the treatment with 4 lM within the same time level (4 or 16 h), and #p < 0.05 treatment for 16 h compared with the treatment for 4 h at the same concentration of BvvTI (4 or 20 lM).
site residues of B. variegata var. purple TI, but different with B. variegata var. candida TI-3 (reactive site: Arg-Ile) [6]. In this study, we also investigated whether BvvTI contained medicinal properties advantageous to the fields of HIV and tumor therapy. Serpins of both endogenous and exogenous origins have been reported to have anti-HIV-1 activity [27,28]. The current study shows that BvvTI could decrease the activity of HIV-1-RT with a small IC50. Potential mechanism may relate to its antiprotease activity and needs further investigation [27]. BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way, and exerted the
ability to induce apoptotic bodies (Table 2 and Fig. 3). The endemic NPC is a non-lymphomatous, squamous-cell carcinoma with a high mortality in northern Africa, Alaska, and especially in South China and Hong Kong [29]. Our data show that BvvTI could potently inhibit the proliferation of well-differentiated CNE-1 cells, and less potently inhibited the poorly differentiated CNE2 cells and the poorly differentiated HNE-2 cells. In addition to the results that there was no significant growth effect on the normal NP 69 cells, the highly potent inhibition of the high-differentiated NPC cells by BvvTI might lead to a way to establish more specific and effective treatments for NPC patients as one
E.F. Fang et al. / Biochemical and Biophysical Research Communications 396 (2010) 806–811
successful personalized therapy for lung cancer was reported recently [30]. Furthermore, it induced the production of multiple cytokines including IL-1b, IL-2 and TNF-a (Fig. 4). This finding is reminiscent of observations on the association between cytokine-inducing and anti-tumor activities of other proteins [31,32]. In a variety of animal cancer models, INF-c, IL-1b, IL-2 and TNF-a act as crucial anti-tumor factors, to suppress the proliferation and reduce the invasive potential of human carcinoma cells by stimulating immune cells including T cells and natural killer cells [33–35]. Our observations on cytokine-inducing activities of BvvTI provide some insight into its underlying anti-tumor mechanism. 5. Conclusion In conclusion, we carried out isolation and molecular characterization of a new Kunitz TI from seeds of B. variegata var. variegata. To the best of our knowledge, this is the first report on a Bauhinia trypsin inhibitor with the abilities to induce the production of multiple cytokines and selectively inhibit the proliferation of nasopharyngeal carcinoma CNE-1 cells. The demonstration of the various aforementioned biological activities highlights the potential medicinal applications of BvvTI. Acknowledgments We thank Dr. Carol P.Y. Lau, Prof. Lawrence Ramsden and Prof. Richard M.K. Saunders from School of Biological Sciences, the University of Hong Kong, for their kind support of the project. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.04.140. References [1] C.P.Y. Lau, L. Ramsden, R.M.K. Saunders, Hybrid origin of ‘‘Bauhinia blakeana” (Leguminosae: Caesalpinioideae), inferred using morphological reproductive, and molecular data, Am. J. Bot. 92 (2005) 525–533. [2] C.P.Y. Lau, The Six Common Bauhinia Species in Hong Kong, Porcupine, vol. 28, Department of Ecology & Biodiversity, The University of Hong Kong, Hong Kong, 2003. [3] M.S. Ali, I. Azhar, Z. Amtul, V.U. Ahmad, K. Usmanghani, Antimicrobial screening of some Caesalpiniaceae, Fitoterapia 70 (1999) 299–304. [4] R.N. Yadava, V.M.S. Reddy, Anti-inflammatory activity of a novel flavonol glycoside from the Bauhinia variegata Linn, Nat. Prod. Res. 17 (2003) 165–169. [5] A.P. Araujo, D. Hansen, D.F. Vieira, C. Oliveira, L.A. Santana, L.M. Beltramini, C.A. Sampaio, M.U. Sampaio, M.L. Oliva, Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies, Biol. Chem. 386 (2005) 561–568. [6] L. Di Ciero, M.L. Oliva, R. Torquato, P. Kohler, J.K. Weder, J. Camillo Novello, C.A. Sampaio, B. Oliveira, S. Marangoni, The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds, J. Protein Chem. 17 (1998) 827–834. [7] A.F. de Souza, R.J. Torquato, A.S. Tanaka, C.A. Sampaio, Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI, Biol. Chem. 386 (2005) 1185–1189. [8] T.B. Ng, S.K. Lam, W.P. Fong, A homodimeric sporamin-type trypsin inhibitor with antiproliferative, HIV reverse transcriptase-inhibitory and antifungal activities from wampee (Clausena lansium) seeds, Biol. Chem. 384 (2003) 289– 293. [9] C.A. Sampaio, M.L. Oliva, M.U. Sampaio, I.F. Batista, N.R. Bueno, A.S. Tanaka, E.A. Auerswald, H. Fritz, Plant serine proteinase inhibitors. Structure and biochemical applications on plasma kallikrein and related enzymes, Immunopharmacology 32 (1996) 62–66. [10] C.I. Cagliari, F.P. De Caroli, A.M. Nakahata, M.S. Araujo, C.R. Nakaie, M.U. Sampaio, C.A.M. Sampaio, M.L.V. Oliva, Action of Bauhinia bauhinioides synthetic peptides on serine proteinases, Biochem. Biophys. Res. Commun. 311 (2003) 241–245.
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Babu, Purification and biochemical characterization of a serine proteinase inhibitor from Derris trifoliata Lour. seeds: insight into structural and antimalarial features, Phytochemistry 70 (2009) 703–712. [26] K.Y. Ee, J. Zhao, A.U. Rehman, S.O. Agboola, Purification and characterization of a Kunitz-type trypsin inhibitor from Acacia victoriae Bentham seeds, J. Agric. Food Chem. 57 (2009) 7022–7029. [27] L. Shapiro, G.B. Pott, A.H. Ralston, Alpha-1-antitrypsin inhibits human immunodeficiency virus type 1, FASEB J. 15 (2001) 115–122. [28] T.B. McNeely, D.C. Shugars, M. Rosendahl, C. Tucker, S.P. Eisenberg, S.M. Wahl, Inhibition of human immunodeficiency virus type 1 infectivity by secretory leukocyte protease inhibitor occurs prior to viral reverse transcription, Blood 90 (1997) 1141–1149. [29] W.I. Wei, J.S. Sham, Nasopharyngeal carcinoma, Lancet 365 (2005) 2041–2054. [30] T.S. Mok, Y.L. Wu, S. Thongprasert, C.H. Yang, D.T. Chu, N. Saijo, P. Sunpaweravong, B. Han, B. Margono, Y. 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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Bauhinia variegata var. variegata trypsin inhibitor: From isolation to potential medicinal applications Evandro Fei Fang a, Jack Ho Wong a, Clara Shui Fern Bah b, Peng Lin a, Sai Wah Tsao c, Tzi Bun Ng a,* a
School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China Department of Food Science, Division of Sciences, University of Otago, New Zealand c Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Sassoon Road, Pokfulam, Hong Kong SAR, China b
a r t i c l e
i n f o
Article history: Received 13 April 2010 Available online 8 May 2010 Keywords: Bauhinia variegata var. variegata Proteinase inhibitors Cytokines HIV Nasopharyngeal carcinoma
a b s t r a c t Here we report for the first time of a new Kunitz-type trypsin inhibitor (termed BvvTI) from seeds of the Camel’s foot tree, Bauhinia variegata var. variegata. BvvTI shares the same reactive site residues (Arg, Ser) and exhibits a homology of N-terminal amino acid sequence to other Bauhinia protease inhibitors. The trypsin inhibitory activity (Ki, 0.1 109 M) of BvvTI ranks the highest among them. Besides anti-HIV1 reverse transcriptase activity, BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way. This may partially be contributed by its induction of cytokines and apoptotic bodies. These results unveil potential medicinal applications of BvvTI. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Bauhinia variegata (Camel’s foot tree), a species of flowering plant belonging to the family Leguminosae, has long been cultivated as an ornamental tree in Hong Kong [1]. It is one of the three common Bauhinia tree species in Hong Kong, the other two being Bauhinia purpurea (Purple camel’s foot) and Bauhinia blakeana Dunn (the Hong Kong Orchid Tree) [2]. Studies on the varieties of B. variegata have indicated that a number of medicinal components can be purified from the plant. Its stem displays antibacterial, antifungal, as well as anti-tumor activities while its roots have antiinflammatory activity [3,4]. As legume seeds usually have a high content of serine protease inhibitors, it is therefore not surprising that serine protease inhibitors have been identified in different varieties of B. variegata [5–7]. Serine protease inhibitors (serpins) from leguminous seeds inactivate several enzymes, and are classified into families including Kunitz trypsin inhibitors (TIs), Bowman–Birk TI and others, depending on their molecular mass and cysteine content [8,9]. Kunitz-type TIs are characterized by their ability to inhibit proteases that are involved in physiological events such as coagulation, inflammation, and cancer [9]. Due to the involvement of proteases in several pathological events, characterizing the structure and function of new protease inhibitors may identify important tools for the fields of disease mechanism exploration to drug design [10,11]. * Corresponding author. Address: Room 302B, BMSB, The Chinese University of Hong Kong, Hong Kong, China. Fax: +852 26035123. E-mail addresses: [email protected], [email protected] (T.B. Ng). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.140
Here, we for the first time describe the isolation, biochemical and functional properties of a TI, BvvTI, from the seeds of a local variety of B. variegata (var. variegata). The results show BvvTI has significant anti-HIV-1 reverse transcriptase and anti-nasopharyngeal carcinoma CNE-1 properties and unveils its potential medicinal applications. 2. Materials and methods 2.1. Protein purification Mature seeds (250 g) of B. variegata var. variegata were harvested on the campus of the Chinese University of Hong Kong (CUHK) and authenticated by Prof. Shiu Ying Hu, honorary Professor of Chinese Medicine, CUHK. The supernatant derived from centrifugation of the seed homogenate was first loaded onto a SPSepharose column (GE Healthcare, UK). The adsorbed faction was then loaded on a Mono S 5/50 GL column, followed on a Superdex 75 10/300 GL column, using an AKTA Explorer 100 chromatography system (GE Healthcare, UK). The purified BvvTI in peak BVSUPI was dialyzed extensively, freeze-dried, and stored at 20 °C for further studies. 2.2. Molecular mass and N-terminal amino acid sequence determination Both sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for molecular
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weight determination were carried out according to methods described in Ref. [12]. N-terminal amino acid sequence analysis was performed using a HP 1000A Edman degradation unit and a HP 1000 HPLC system [12]. The percentage of sequence identity with TIs from related legumes was determined using BCM search launcher, the software ClustalX 1.83, and the on line BOXSHADE 3.21 server [13].
against trypsin was tested and compared with a negative control. Reagents used included phenylglyoxal (PG, for arginine resides modification) [20], phenylmethylsulfonyl fluoride (PMSF, for serine modification), 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB, for reduction of the thiol groups), sodium borohydride (SB, for lysine modification), and N-acetylimidazole (NA, for tyrosine residues modification) [19].
2.3. Enzymatic analysis
2.6. Assay of HIV-1 reverse transcriptase (HIV-1-RT) inhibitory activity
2.3.1. With specific synthetic substrate The assay was done by using a spectrophotometric method with some modifications [12,14,15]. Firstly, different doses of 20 lL BvvTI in 50 mM Tris–HCl (pH 7.6, final concentrations from 0.125, 0.25, 0.5, 0.75, to 1 nM) were incubated with 40 lL 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 of each tube was measured by adding 1.44 mL N-a-benzoyl-L-arginine ethyl ester (BAEE, two different final concentrations from 0.5 to 1 mM) as substrate. After immediately mixing by inversion, the increase in A253 was recorded for 5 min (37 °C). Reaction without the addition of sample was used as a control:
The assay was performed using an HIV-1-RT (recombinant) ELISA kit, following the instructions of the manufacturer (Boehringer Mannheim, Germany) [12]. Pinto bean lectin with anti-HIV-1-RT activity was chosen as a positive control [21].
Calculation : BAEE units=mg trypsin inhibitor ¼ ðDA253=min Positive DA253=min Sample TestedÞ=½ð0:001Þð0:04Þ ðsampleÞ where (0.001) = the change in A253/min per unit of trypsin at pH 7.6 and 37 °C in a 1.5 mL reaction mixture; (0.04) or (sample) = solid (mg) of trypsin or sample used in the reaction. In addition, 1 U 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 37 °C. Chymotrypsin inhibitory activity was determined using the same assay, but by adding more BvvTI (final concentrations from 12.5, 25, 50, 75, to 100 nM) and using N-benzoyl-L-tyrosine ethyl ester (BTEE) as the substrate instead. One a-chymotrypsin unit hydrolyzes 1.0 lmol of BTEE per minute at pH 7.6 and 37 °C. Inhibition constants (Ki) of BvvTI against both trypsin and achymotrypsin were determined as described elsewhere [16]. The Ki value was estimated by using Sigma Plot 10.0, Enzyme Kinetics Module 1.3 (Systat Software Inc., San Jose, USA). 2.3.2. With casein as the substrate In addition, 1% casein was used as substrate for the description of a dose-dependent relationship between BvvTI and residual trypsin activity as mentioned previously [15,17]. Soybean trypsin inhibitor from USB Corporation was used as a positive control.
2.7. Assay of antiproliferative and apoptosis-inducing activity All seven cell lines including six tumor cell lines (human hepatocellular carcinoma Hep G2, human breast tumor MCF-7, mouse leukemia L1210, and three human nasopharyngeal carcinoma CNE-1, CNE-2, and HNE-2 cell lines) and one transformed normal human nasopharyngeal epithelial cell line NP 69 were cultured as previously described [12]. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test was used to monitor inhibition of cell growth following the method reported elsewhere [22]. Apoptosis was observed by chromatin staining with Hoechst 33342 (Sigma, USA) by using the cell line CNE-1 as a paradigm. 2.8. Assay of cytokine-inducing activity Splenocytes from male BALB/c mice were prepared for the assay. After extraction of total RNA using TRIZOLÒ (Invitrogen, USA), changes of cytokine expression at the mRNA level was measured by using a reverse transcriptase PCR kit (GeneAmpÒ, USA). Cytokines including tumor necrosis factor alpha (TNF-a), interleukin-1b (IL-1b), interleukin-2 (IL-2), and interferon-gamma (INF-c) were measured [23]. 2.9. Statistical analysis Results were taken from three independent experiments performed in triplicate, and data were expressed as means ± standard deviation (SD). The median inhibition concentration (IC50) was determined using SPSS 11.0.1 statistical software (SPSS Inc., Chicago, USA). For between-group comparisons, Student’s t-test or one-way ANOVA was used where appropriate and differences with p values of 0.05 or less were considered to be statistically significant. 3. Results
2.4. Thermal and pH stability 3.1. Isolation of BvvTI Thermal and pH stability assays were conducted as previously reported [12,18]. Remaining trypsin inhibitory activity was calculated as percent of the value at 30 °C or pH 7. 2.5. Reactive sites determination The effect of the reducing agent dithiothreitol (DTT) on the stability of BvvTI was examined following the method of Ramos et al. [18]. Soybean TI was used as a positive control. Chemical modification of amino acid residues on the trypsin inhibitory activity of BvvTI was carried out as previously described [16,19]. After treatment with different reagents, the residual inhibitory activity
BvvTI was purified using the method shown in Fig. 1A and Supplementary Fig. 1. The crude seed extract was first run on a SP-Sepharose column, followed by FPLC on a Mono S column. Five major adsorbed peaks (Supplementary Fig. 1A) were obtained. The inhibitor-containing peak, BV-MSIV, was subsequently subjected to size exclusion chromatography on Superdex 75, and purified BvvTI was acquired from peak BV-SUPI. The purity of the BvvTI was confirmed by presence of a sharp single peak in both FPLC-gel filtration on Superdex 75 (data not shown) and mass spectrometry (Fig. 1C), and a single band in SDS–PAGE (Fig. 1B lane 4). Data in Table 1 and Fig. 1 tracked the purification steps of BvvTI.
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Fig. 1. Purification of BvvTI. (A) Schematic representation of the chromatographic steps used to purify BvvTV. (B) SDS–PAGE showing purity and molecular weight of BvvTI. Lane 1, B. variegata var. variegata crude extract (20 lg); lane 2, fraction adsorbed on SP-Sepharose (30 lg); lane 3, peak BV-MSIV from FPLC-Mono-S column (20 lg); lane 4, peak BV-SUPI from Superdex 75 column (20 lg); and lane 5, molecular weight marker from GE Healthcare. (C) The MALDI-TOF mass spectrometry results of BvvTI showing a peak of 21,096 Da.
Table 1 Summary of purification of BvvTI from 250 g mature seeds of Bauhinia variegata var. variegata. Sample
Step (peak)
Total protein (mg)
Specific trypsin inhibitory activity (BAEE units/mg)
Purification fold*
Yield (%)
BvvTI
Water extract Sp-Sepharose Mono S (BV-MSIV) Superdex 75 (BV-SUPI)
10,200 3824 692 429
294 890 3320 5865
1.0 3.0 11.3 20.0
100 37.5 6.8 4.2
*Purification fold equals specific trypsin inhibitory activity of the chromatographic fraction divided by specific trypsin inhibitory activity of the water extract fraction.
3.2. Biological and enzymatic activities of BvvTI
3.4. Anti-HIV-1-RT activity
SDS–PAGE of acquired BvvTI displayed only a single band which revealed that the protein was homogeneous and was a single 21-kDa polypeptide chain in accordance with the MALDI-TOF MS results (Fig. 1B and C). The sequence of the first 20 N-terminal amino acids of BvvTI is as follows: DTLLD TDGEV VRNSG GRYYI. As shown in Supplementary Table 1, in comparison with Kunitz TIs from other Bauhinia species, a pronounced homology was observed. By using specific synthetic substrates, the inhibitory activities of BvvTI towards trypsin and a-chymotrypsin were 5865 BAEE units/ mg and 43 BTEE units/mg, respectively (Table 1). In addition, a relationship between BvvTI dose and residual trypsin or a-chymotrypsin activity is shown in Supplementary Fig. 2 when casein was used as a substrate. Lineweaver–Burk double reciprocal plots (Supplementary Fig. 3A and C) and Dixon plots (Supplementary Fig. 3B and D) showed a non-competitive-type inhibitory activity of BvvTI against both trypsin and a-chymotrypsin and with Kis of 0.1 109 M and 250.5 109 M, respectively.
In vitro ELISA results revealed that BvvTI could inhibit the activity of HIV-1-RT with an IC50 value of 6.4 lM (Supplementary Fig. 4). Results of the positive control, the pinto bean lectin, yielded an IC50 value of 2.5 lM which compares well with previously reported values [21].
3.3. Stability of BvvTI and its potential reactive site residues Fig. 2A, B and C show the thermal, pH and chemical stabilities of BvvTI. The trypsin inhibitory activity of BvvTI was stable up to 50 °C and in buffers with pH 4–12. Furthermore, BvvTI was able to withstand reducing conditions of 1 mM DTT for 0.5 h, but there was a steady loss of activity to 89% and 27% of the initial activity when the DTT concentration was augmented to 10 mM and 100 mM, respectively (Fig. 2C). As shown in Fig. 2D, modifications of both arginine residues and serine residues brought about serious reduction (about 75% and 55%, respectively) of its trypsin inhibitory activities. However, no significant loss of activity was observed after modification of thiol groups, lysine or tyrosine residues.
3.5. Anti-tumor and cytokine-inducing activities As listed in Table 2, the MTT assay revealed that BvvTI exerted a specific antiproliferative activity on CNE-1 cells in a time-dependent manner. There was no significant effect on other tumor cells and the normal cell line NP 69 even after 48 h of exposure. CNE1 was further chosen to study the apoptosis-inducing ability of BvvTI. Fig. 3 shows the morphological changes of CNE-1 cells when the BvvTI concentration was increased from 0 to 100 lM, and the reduction of cell numbers and apoptotic bodies were noticed accordingly (Fig. 3). BvvTI differentially regulates the mRNA expression of cytokines including IL-1b, IL-2, TNF-a, and INF-c. As shown in Fig. 4, BvvTI significantly increased the transcriptional levels of IL-1b, IL-2 and TNF-a in a dose-dependent effect at a specified time point. However, the cytokine-inducing ability towards INF-c was not significant. 4. Discussion BvvTI was isolated by employing a simple liquid chromatography method with an acceptable harvest rate of approximately 5% (Table 1) similar to that achieved using other methods [6]. The biological characteristics of BvvTI demonstrate similarity with other Bauhinia Kunitz-type TIs, including an identical molecular weight (Fig. 1) and a high degree of homology of N-terminal amino acid sequence (Supplementary Table 1) [6,7,24].
809
A 120
B 120
Residual trypsin inhibitory activity (%)
Residual trypsin inhibitory activity (%)
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100
* *
80
60
*
40
20
* *
0
100
* 80
*
60
20 0
0
20
40
60
80
100
120
0
2
4
6
Temperature
D BvvTI
Soybean TI
* *
80
60
*
40
*
20
0
1
0
10
100
DTT (mM)
Residual trypsin inhibitory activity (%)
Residual trypsin inhibitory activity (%)
C 120 100
8
10
12
14
pH
120
100
80
*
60
40
*
20
0
B B G TI SF NA TN +S Bvv TI + P + PM TI + + D vvTI v v I I v v T T B B B Bv v Bvv
Fig. 2. Stability of BvvTI and its potential reactive site residues. (A) Thermal stability was tested after BvvTI (50 lM in Tris–HCl buffer) had been exposed to 0–100 °C for 30 min. (B) pH stability of BvvTI was measured after dissolving the lyophilized protein in buffers of different values to a final concentration of 50 lM, followed by incubation for 0.5 h at 37 °C. (C) BvvTI were incubated with dithiothreitol (DTT) at different final concentrations (0, 1, 10, and 100 mM) for 30 min at 37 °C. Soybean TI was used as a positive control. (D) Chemical modification of amino acid residues on the trypsin inhibitory activity of BvvTI. Reagents in details were in Section 2.5. Residual trypsin inhibitory activity in all (A), (B) (C) and (D) was measured by using BAEE as substrate. Means ± SD of three independent experiments are shown. *p < 0.05 versus the residual activity at 30 °C (A) or pH 7 (B) or negative control (C, D), respectively.
Table 2 Antiproliferative activity of BvvTI on different tumor cell lines. Tumor cell line
Hep G2 MCF-7 L1210 CNE-1 CNE-2 HNE-2 NP 69 (normal)
IC50 (lM) 24 h
48 h
225.36 ± 27.44 – – 54.27 ± 4.29 – – –
87.89 ± 5.64* – – 21.37 ± 2.36* 354.70 ± 24.89* – –
Values of IC50 represent means ± SD of three determinations using the MTT assay. –, antiproliferative activity was undetectable at BvvTI concentrations above 1 mM. * p < 0.05 compared with IC50 (24 h) of BvvTI for the same cell line.
Results obtained by using specific synthetic substrates (BAEE and BTEE) and the non-specific substrate casein, manifest a significant trypsin inhibitory activity and a slighter antiproteolytic activity against chymotrypsin (Table 1, and Supplementary Figs. 2 and 3). This was to some extent similar to other Kunitz TIs [6,7,18,24– 26]. To the best of our knowledge, the Ki (0.1 109 M) of BvvTI to-
wards trypsin is so far the highest among Bauhinia serine protease inhibitors [5–7,9,24]. For example, the Ki values towards trypsin for BvcTI (B. variegata var. candida TI) and BvlTI (B. variegata var. lilac TI) are 6.9 and 1.2 nM, respectively [6]. A much bigger Ki value observed for a-chymotrypsin, unveiled a higher affinity between BvvTI and trypsin, in accordance with previous literature on Bauhinia [6,7,9,24]. Several structural features are conserved in most Kunitz-type inhibitors, including molecular mass (20 kDa), four cysteine residues arranged in two disulfide bridges, and the single reactive site (Arg-Ser or Arg-Ile) [5,6]. However, there are some exceptions. For instance, two 18-kDa protease inhibitors from Bauhinia bauhinioides are devoid of disulfide bridges and with different reactive site [5]. For BvvTI, DTT treatment (break down the disulfide bonds) resulted in a gradual loss of trypsin inhibitory activity, which sheds light on the importance of the disulfide bond to its trypsin/a-chymotrypsin inhibitory activities [18]. Furthermore, chemical modification studies were carried out to identify the amino acids responsible for the inhibitory activity of BvvTI against trypsin. The results (Fig. 2D) manifest that both Arg and Ser residues present in the reactive site play a significant role in its antiproteolytic activity towards trypsin, which was coincidence with the reactive
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Fig. 3. Dose-escalation study of BvvTI-induced nuclear morphological changes in CNE-1 cells. After treatment with different doses of BvvTI including (A) 0 lM (control), (B) 10 lM, (C) 50 lM, and (D) 100 lM for 24 h, CNE-1 cells were stained with 1 lg/mL Hoechst 33342 for 10 min and the morphological changes of cells were examined by fluorescence microscopy. Row 1, brightfield images; row 2, fluorescent images; row 3, merged images between row 1 and row 2; row 4, 5-fold zoom of merged images (row 3) as indicated by thin arrows. Typical apoptotic bodies in D-4 are indicated by arrows. Bar in merged image indicates 20 lm.
Fig. 4. Induction of cytokine mRNA expression by BvvTI. Mouse splenocytes were treated with BvvTI at final concentrations of 4 or 20 lM for 4–20 h. Its cytokine-inducing activity was measured by RT-PCR. The experiment was performed in triplicate and one set of typical results is shown (A). Lane 1, DNA ladder; lane 2, control (splenocytes incubated for 16 h without any treatment); lane 3, splenocytes treated with 4 lM BvvTI for 4 h; lane 4, 20 lM BvvTI for 4 h; lane 5, 4 lM BvvTI for16 h; and lane 6, 20 lM BvvTI for 16 h. Fig. (B) RT-PCR results from three independent experiments. Band intensity was measured using Alphaimager 2200 in which each cytokine mRNA expression was normalized relative to the GAPHD expression levels. *p < 0.05 compared with control, p < 0.05 treatment with 20 lM compared with the treatment with 4 lM within the same time level (4 or 16 h), and #p < 0.05 treatment for 16 h compared with the treatment for 4 h at the same concentration of BvvTI (4 or 20 lM).
site residues of B. variegata var. purple TI, but different with B. variegata var. candida TI-3 (reactive site: Arg-Ile) [6]. In this study, we also investigated whether BvvTI contained medicinal properties advantageous to the fields of HIV and tumor therapy. Serpins of both endogenous and exogenous origins have been reported to have anti-HIV-1 activity [27,28]. The current study shows that BvvTI could decrease the activity of HIV-1-RT with a small IC50. Potential mechanism may relate to its antiprotease activity and needs further investigation [27]. BvvTI could significantly inhibit the proliferation of nasopharyngeal cancer CNE-1 cells in a selective way, and exerted the
ability to induce apoptotic bodies (Table 2 and Fig. 3). The endemic NPC is a non-lymphomatous, squamous-cell carcinoma with a high mortality in northern Africa, Alaska, and especially in South China and Hong Kong [29]. Our data show that BvvTI could potently inhibit the proliferation of well-differentiated CNE-1 cells, and less potently inhibited the poorly differentiated CNE2 cells and the poorly differentiated HNE-2 cells. In addition to the results that there was no significant growth effect on the normal NP 69 cells, the highly potent inhibition of the high-differentiated NPC cells by BvvTI might lead to a way to establish more specific and effective treatments for NPC patients as one
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successful personalized therapy for lung cancer was reported recently [30]. Furthermore, it induced the production of multiple cytokines including IL-1b, IL-2 and TNF-a (Fig. 4). This finding is reminiscent of observations on the association between cytokine-inducing and anti-tumor activities of other proteins [31,32]. In a variety of animal cancer models, INF-c, IL-1b, IL-2 and TNF-a act as crucial anti-tumor factors, to suppress the proliferation and reduce the invasive potential of human carcinoma cells by stimulating immune cells including T cells and natural killer cells [33–35]. Our observations on cytokine-inducing activities of BvvTI provide some insight into its underlying anti-tumor mechanism. 5. Conclusion In conclusion, we carried out isolation and molecular characterization of a new Kunitz TI from seeds of B. variegata var. variegata. To the best of our knowledge, this is the first report on a Bauhinia trypsin inhibitor with the abilities to induce the production of multiple cytokines and selectively inhibit the proliferation of nasopharyngeal carcinoma CNE-1 cells. The demonstration of the various aforementioned biological activities highlights the potential medicinal applications of BvvTI. Acknowledgments We thank Dr. Carol P.Y. Lau, Prof. Lawrence Ramsden and Prof. Richard M.K. Saunders from School of Biological Sciences, the University of Hong Kong, for their kind support of the project. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.04.140. References [1] C.P.Y. Lau, L. Ramsden, R.M.K. Saunders, Hybrid origin of ‘‘Bauhinia blakeana” (Leguminosae: Caesalpinioideae), inferred using morphological reproductive, and molecular data, Am. J. Bot. 92 (2005) 525–533. [2] C.P.Y. Lau, The Six Common Bauhinia Species in Hong Kong, Porcupine, vol. 28, Department of Ecology & Biodiversity, The University of Hong Kong, Hong Kong, 2003. [3] M.S. Ali, I. Azhar, Z. Amtul, V.U. Ahmad, K. Usmanghani, Antimicrobial screening of some Caesalpiniaceae, Fitoterapia 70 (1999) 299–304. [4] R.N. Yadava, V.M.S. Reddy, Anti-inflammatory activity of a novel flavonol glycoside from the Bauhinia variegata Linn, Nat. Prod. Res. 17 (2003) 165–169. [5] A.P. Araujo, D. Hansen, D.F. Vieira, C. Oliveira, L.A. Santana, L.M. Beltramini, C.A. Sampaio, M.U. Sampaio, M.L. Oliva, Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies, Biol. Chem. 386 (2005) 561–568. [6] L. Di Ciero, M.L. Oliva, R. Torquato, P. Kohler, J.K. Weder, J. Camillo Novello, C.A. Sampaio, B. Oliveira, S. Marangoni, The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds, J. Protein Chem. 17 (1998) 827–834. [7] A.F. de Souza, R.J. Torquato, A.S. Tanaka, C.A. Sampaio, Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI, Biol. Chem. 386 (2005) 1185–1189. [8] T.B. Ng, S.K. Lam, W.P. Fong, A homodimeric sporamin-type trypsin inhibitor with antiproliferative, HIV reverse transcriptase-inhibitory and antifungal activities from wampee (Clausena lansium) seeds, Biol. Chem. 384 (2003) 289– 293. [9] C.A. Sampaio, M.L. Oliva, M.U. Sampaio, I.F. Batista, N.R. Bueno, A.S. Tanaka, E.A. Auerswald, H. Fritz, Plant serine proteinase inhibitors. Structure and biochemical applications on plasma kallikrein and related enzymes, Immunopharmacology 32 (1996) 62–66. [10] C.I. Cagliari, F.P. De Caroli, A.M. Nakahata, M.S. Araujo, C.R. Nakaie, M.U. Sampaio, C.A.M. Sampaio, M.L.V. Oliva, Action of Bauhinia bauhinioides synthetic peptides on serine proteinases, Biochem. Biophys. Res. Commun. 311 (2003) 241–245.
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