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A protein with antiproliferative, antifungal and HIV-1 reverse transcriptase inhibitory activities from caper (Capparis spinosa) seeds.

Abstract

A protein exhibiting an N-terminal amino acid sequence with some similarity to imidazoleglycerol phosphate synthase was purified from fresh Capparis spinosa melon seeds. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, cation exchange chromatography on SP-Sepharose, and finally gel filtration by fast protein liquid chromatography on Superdex 75. The protein was adsorbed using 20 mM Tris-HCl buffer (pH 7.4) and desorbed using 1 M NaCl in the starting buffer from the DEAE-cellulose column and SP-Sepharose column. The protein demonstrated a molecular mass of 38 kDa in gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it was monomeric. The protein inhibited proliferation of hepatoma HepG2 cells, colon cancer HT29 cells and breast cancer MCF-7 cells with an [IC.sub.50] of about 1, 40 and 60 [mu]M, respectively. It inhibited HIV-1 reverse transcriptase with [IC.sub.50] of 0.23 [mu]M. It inhibited mycelial growth in the fungus, Valsa mali. It did not exhibit hemagglutinating, ribonuclease, mitogenic or protease inhibitory activities.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Antiproliferative protein; Caper (Capparis spinosa)

Introduction

Capparis spinosa is a plant belonging to Family Capparaceae. Different parts of this plant exhibit biological activity. The bud extract suppressed the replication of Herpes simplex virus type 2 and increased the expression of pro-inflammatory cytokines including interleukin-12, interferon-[gamma] and tumor necrosis factor-[alpha] (Arena et al., 2008). Flavanols and hydroxycinnamic acid are antioxidants present in the buds (Panico et al., 2005). p-Methoxybenzoic acid is an antihepatotoxic component in the methanolic fraction of the aqueous extract of the plant (Gadgoli and Mishra, 1999). The aqueous extract exhibited anti-hyperglycemic (Eddouks et al., 2004) and hypolipidemic (Eddouks et al., 2005) activities. Other activities included antiviral, immunomodulatory (Arena et al., 2008), chondrocyte protective (Panico et al., 2004), anti-allergeric, antihistaminic (Trombetta et al., 2005), antifungal (Ali-Shtaych and Abu Ghdeib, 1999), anti-Leishmania (Jacobson and Schlein, 1999) and antimicrobial (Mahasneh, 2002) activities, as well as inhibitory effect on fibroblast proliferation and type 1 collagen production in progressive systemic sclerosis (Cao et al., 2008).

In view of the scarcity of information on the proteinaceous constituents of C. spinosa, the purpose of the present study was undertaken to isolate an antiproliferative protein from the seeds of this melon. A preliminary experiment indicated that the seed extract had antiproliferative activity toward tumor cells.

Materials and methods

Isolation of protein

Fresh Caper (C. spinosa) melons (3.6 kg) were purchased from a local vendor. The seeds (580 g) collected from the melons were extracted by homogenizing in distilled water using a Waring blender. Following centrifugation at 20.000g for 30 min at 4[degrees]C, the supernatant was collected. Tris-HCl buffer (2 M, pH 7.4) was added to the supernatant until the final concentration of Tris-HCl reached 20 mM. The supernatant was then applied on a 5 cm (diameter) x 9 cm (length) column of DEAE-cellulose (Sigma, St. Louis, Missouri, USA). Unadsorbed proteins were eluted with 20 mM Tris-HCl buffer (pH 7.4). Adsorbed proteins were eluted stepwise, first with 0.1 M NaCl and then with 1 M NaCl added to the 20 mM Tris-HCl buffer. The fraction eluted with 1 M NaCl was taken, dialyzed extensively against water and then subjected to ion exchange chromatography on a 5 cm (diameter) x 4.5 cm (length) column of SP-Sepharose (GE Healthcare, Hong Kong, China). After removal of unadsorbed proteins with 20 mM Tris-HCl buffer (pH 7.4), adsorbed proteins were eluted with 1 M NaCl added to the 20 mM Tris-HCl buffer. The unadsorbed fraction was saved and dialyzed extensively against water and lyophilized before re-dissolving and chromatography on a Superdex 75 HR 10/30 column in 200 mM [NH.sub.4][HCO.sub.3] (pH 7) (GE Healthcare) using an AKTA Purifier (GE Healthcare). The second fraction collected represented purified antiproliferative protein.

Sodium dodecyl sulfate--polyacrylamide gel electrophoresis

The purified protein was subjected to sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) for molecular mass determination in accordance with the procedure described (Laemmli and Favre, 1973).

Determination of native molecular weight by gel filtration

Gel filtration on a fast protein liquid chromatography (FPLC)-Superdex 75 column, which had been calibrated with molecular mass markers, including Blue Dextran 2000. aldolase (158 kDa), BSA (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), myoglobulin (17.6 kDa), ribonuclease A (13.7 kDa), aprotinin (6.5 Da) and vitamin B12 (1.3 kDa) (GE Healthcare), was conducted to determine the molecular mass of the protein.

N-terminal amino acid sequence determination

The N-terminal amino acid sequence of the protein was determined by using a Hewlett-Packard HP G1000A Edman degradation unit and an HP 1000 high-performance liquid chromatography (HPLC) system (Wang et al., 2000).

Assay of antiproliferative activity

The assay of the antiproliferative activity of the isolated protein was carried out by testing its inhibition on the growth of the human hepatoma cell line HepG2, the human colon cancer cell line HT29 and the human breast cancer cell line MCF-7. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, in a humidified atmosphere of 5% [CO.sub.2] at 37[degrees]C. The cells were cultured in the flask and trypsinized for counting using a Thoma's hemacytometer. The cell number was adjusted to 5 x [10.sup.4] cells/ml by addition of the medium. The cells (5 x [10.sup.3]cells/100 [mu]l/well) were seeded in a 96-well culture plate and serial dilutions of a solution of the isolated protein or doxorubicin (as positive control) in 100 [mu]l medium were added. After incubation of the cells at 37[degrees]C in a humidified atmosphere of 5% [CO.sub.2] for 48 h, the cells were harvested and stained with MTT. The absorbances of the samples at 595 nm were determined using a microtiter plate (ELISA) reader and were directly correlated to the level of its antiproliferative activity. The inhibitory activity of the isolated protein was calculated as percent of cell viability compared to a control without the sample. All reported values are the means of triplicate samples (Wang et al., 2000).

Assay of hemagglutinating activity

This assay was conducted because lectins exhibit antiproliferative activity (Wong and Ng, 2003). In the assay for lectin (hemagglutinating) activity, a serial twofold dilution of the protein solution or solution of Concanavalin A (as positive control) in microtiter U-plates (50 [mu]l) was mixed with 50 [mu]l of a 2% suspension of rabbit red blood cells in phosphate-buffered saline (pH 7.2) at 20[degrees]C. The results were read after about 1 h, when the blank had fully sedimented. The hemagglutination titer, defined as the reciprocal of the highest dilution exhibiting hemagglutination, was reckoned as one hemagglutination unit. Specific activity is the number of hemagglutination units per milligram protein (Wang et al., 2000).

Assay of mitogenic activity in mouse splenocytes

This assay was performed since proteins with antiproliferative activity may elicit the mitogenic response from spleen cells (Wong and Ng, 2003; Ho et al., 2004). The assay of mitogenic activity was performed as described in Wang et al. (2002). Splenocytes were isolated from BALB/c mice. The cells were diluted with RPMI medium containing 10% fetal bovine serum and then seeded (2 x [10.sup.6] cells/0.1 ml/well) in 96-well micro-plates. The protein or Concanavalin A (as positive control) was then added at various concentrations. Cells cultured in the absence of the lectin/isolated protein served as control. The cells were incubated at 37[degrees]C in a humidified atmosphere of 5% carbon dioxide for 72 h. During the last 24 h, the cells in one well were pulsed with 0.5 [mu]Ci [[.sup.3]) H-methyl]thymidine (specific activity 5 [mu]Ci/mmol, GE Healthcare) in 10 [mu]l and were then harvested onto a glass fiber filter using a cell harvester. The radioactivity was determined using a Beckman scintillation counter. The proliferative (mitogenic) response was expressed as mean counts per minute (cpm).

Assay of ribonuclease activity

The isolated protein was assayed for this activity since some ribonucleases have antiproliferative activity (Ye et al., 2000). The activity of the purified protein toward yeast tRNA (Sigma) was assayed by determining the generation of acid-soluble, UV-absorbing species with the method of Wang and Ng (2004). The protein or porcine pancreatic RNase (Sigma) (as positive control) was incubated with 200 [mu]g tRNA in 150 [mu]l of 100 mM MES buffer (pH 5) at 37[degrees]C for 15 min. The reaction was terminated by the introduction of 350 [mu]l of ice-cold 3.4% perchloric acid. After leaving on ice for 15 min, the sample was centrifuged (15,000g, 15 min) at 4[degrees]C. The [OD.sub.260] of the supernatant was read after appropriate dilution. One unit of enzymatic activity is defined as the amount of enzyme that brings about an increase in [OD.sub.260] of one per minute in the acid-soluble fraction per milliliter of reaction mixture under the specified condition. Addition of 200 [mu]g tRNA to RNase was immediately followed by addition of 3.4% perchloric acid. This served as the negative control, Pancreatic RNase (Sigma) was used as a positive control.

Assay of antifungal activity

The isolated protein was assayed for this activity in view of the report showing that antifungal proteins may exhibit antiproliferative activity (Leung and Ng, 2007). The assay for antifungal activity toward the phyto-pathogenic fungi Mycosphaerella arachidicola, Fusarium oxysporum, Helminthosporium maydis, Valsa mali and Rhizoctonia solani was carried out in 100 mm x 15 mm petri plates containing 10 ml of potato dextrose agar. After the mycelial colony had developed, sterile blank paper disks (0.625 cm in diameter) were placed at a distance of 0.5 cm away from the rim of the mycelial colony. An aliquot (15 [mu]l) of the purified protein or nystatin (Sigma) (as positive control) (Leung and Ng, 2007) was added to a disk. The plates were incubated at 23[degrees]C for 72 h until mycelial growth had enveloped the disks containing the control and had formed crescents of inhibition around disks containing samples with antifungal activity (Ye et al., 2000).

Assay of inhibition on HIV-1 reverse transcriptase

The isolated protein was assayed for this activity since some proteins with antiproliferative activity have this activity (Leung and Ng, 2007). The assay of protein for ability to inhibit HIV-1 reverse transcriptase was carried out by using an enzyme-linked immunosorbent assay kit from Boehringer Mannheim (Germany) as described in Collins et al. (1997). The assay takes advantage of the ability of reverse transcriptase to synthesize DNA, starting from the template/primer hybrid poly(a)oligo (dT) 15. Instead of radio-labeled nucleotides, digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into one and the same DNA molecule, which is freshly synthesized by the reverse transcriptase (RT). The detection and quantification of synthesized DNA as a parameter for RT activity follows a sandwich enzyme-linked immunosorbent assay protocol. Biotin-labeled DNA binds to the surface of microtiter plate modules that have been precoated with streptavidin. In the next step an antibody to digoxigen-in, conjugated to peroxidase (anti-DIG-POD), binds to the digoxigenin-labeled DNA. In the final step, the peroxidase substrate is added. The peroxidase enzyme catalyzes the cleavage of the substrate, producing a colored reaction product. The absorbance of the samples at 405 nm can be read using a microtiter plate (ELISA) reader and is directly proportional to the level of RT activity. A fixed amount (4-6 ng) of recombinant HIV-1 reverse transcriptase was used. The inhibitory activity of the purified protein was calculated as percent inhibition as compared to a control without the protein. Brassica campestris lipid transfer protein (Lin et al., 2007) was used as positive control.

Assay of protease inhibitory activity

This assay was performed since some protease inhibitors manifest antiproliferative activity (Lanza et al., 2004; Fernanda Troncoso et al., 2003; Banerji et al., 1998). The inhibitory activities on bovine pancreatic trypsin and bovine-chymotrypsin were determined by measuring the remaining hydrolytic activity toward specific substrates, BAEE and BTEE, respectively, after pre-incubation with PDTI for 10 min. Enzyme-substrate mixtures (100 [mu]l) were added to a solution containing substrate in 50 mM Tris-HCl buffer, pH 8.2, 20 mM [CaCl.sub.2]. The substrate hydrolysis was monitored by measuring absorbance at 253 nm during 1 min. The substrate concentration was 50 [mu]g/ml. Soybean trypsin inhibitor was used as a positive control.

Results and discussion

Ion exchange chromatography of the seed extract on DEAE-cellulose produced a very large unadsorbed fraction (Dl) and two adsorbed fractions (D2 and D3) of approximately the same size. Antiproliferative activity resided only in fraction D3 eluted with 1 M NaCl (Fig. 1). This fraction was separated on SP-Sepharose into a large unadsorbed fraction (P1) with antiproliferative activity and a smaller adsorbed fraction (P2) without activity (Fig. 2). Fraction P1 was subjected to final purification on Superdex 75. Four fractions were obtained. Antiproliferative activity resided in the second fraction S2, which appeared to be much larger than the other fractions (Fig. 3). This fraction demonstrated a single 38-kDa band in SDS-PAGE (Fig. 4) and a single 38-kDa absorbance peak upon re-chromatography on the Superdex 75 gel filtration column (Data not shown). A summary of the yields of the various chromatographic fractions is included in Table 1. The N-terminal sequence of the protein was similar to a partial sequence of imidazoleglycerol phosphate synthase (Table 2). The protein inhibited proliferation of HepG2, HT29 and MCF-7 tumor cells with an [IC.sub.50] of 1, 40 and 60 [mu]M, respectively (Fig. 5, Table 3). It inhibited HIV-1 reverse transcriptase with an [IC.sub.50] of 0.23 [mu]M (Fig. 6, Table 3). The isolated protein inhibited mycelial growth only the case of V. mali (Fig. 7). There was no effect on other fungi including M. arachidicola, F. oxysporum, H. maydis and R. solani. It did not exhibit hemagglutinating, mitogenic, ribonuclease, or protease inhibitory.

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[FIGURE 7 OMITTED]
Table 1. Yields from 580g wet Capparis spinosa seed and at different
stages of purification of Cupparis spinosa antiproliferative protein

Column          Chromatographic fraction  Yield (mg)

-               Crude extract                   1800
DEAE-cellulose  D3                               500
SP-Sepharose    Pl                               300
Superdex 75     S2                               120

Table 2. N-terminal amino acid sequence of Capparis spinosa
antiproliferative protein

                     Residue no.  Sequence    Number of amino acid
                                              residues

Capparis spinosa     1            SYDTQAEAAL  ~340
antiproliferative
protein

Imidazoleglycerol    29           SYD-QAEAAL  260
phosphate synthase,
cyclase subunit,
dehalococcoides
ethenogenes 195


Some proteins are capable of exerting an antiproliferative action on hepatoma HepG2 cells and breast cancer MCF-7 cells. The [IC.sub.50] values of Bowman-Birk trypsin inhibitor from Hokkaido large black soybean (Ho and Ng, 2008), French bean hemagglutinin (Leung et al., 2008), and lipid transfer protein from B. campestris (Lin et al., 2007) are, respectively, 140 and 35, 13 and 6.6, and 5.8 and 35[mu]M. Hence the antiproliferative potency of the isolated protein toward HepG2 cells seems to be relatively high while the potency toward MCF-7 cells is much lower (Table 3).

The protein with antiproliferative activity isolated from C. spinosa melon seeds in the present study is devoid of hemagglulinating, mitogenic, ribonuclease and protease inhibitory activities. This is noteworthy since lectins (Wong and Ng, 2006), ribonucleases (Lam and Ng, 2001), protease inhibitors (Ng et al., 2003) and antifungal proteins (Wang and Ng, 2006) may demonstrate antiproliferative activity toward tumor cells. However, the antifungal activity exhibiting antiproliferative activity of the isolated protein is weak. The protein manifests the highest antiproliferative activity toward hepatoma cells. Variations in the antiproliferative potency of a given protein toward different tumor cell lines have been noted e.g. choriocarcinoma cells are much more sensitive than hepatoma cells to the ribosome inactivating proteins trichosanthin and [alpha]-momorcharin (Tsao et al., 1990).
Table 3. Comparison of biological potencies of Capparis spinosa
antiproliferative protein, doxorubicin and B. campes-tris lipid
transfer protein

               Capparis spinosa   Doxorubicin    B. campestris
               antiproliferative  ([IC.sub.50])  lipid transfer
               protein            ([mu]M)        protein
               ([IC.sub.50])                     ([IC.sub.50)
               ([mu]M)                           ([mu]M)

HepG2                          1             10             5.8

MCF-7                         60              2              35

HT29                          40             10               -

HIV-1                       0.23              -               4

reverse
transcriptase


Asparagus DNase (Wang and Ng, 2001) and shallot antifungal protein (Wang and Ng, 2002) inhibit only one fungal species out of the several tested. The isolated antiproliferative protein also inhibits only V. mali

The isolated protein exhibits a potent HIV-1 reverse transcriptase inhibitory activity with an [IC.sub.50] of 0.23 [mu]M, which is much more potent than many of the anti-HIV-1 natural products (Table 3) (Lin et al., 2007). The mechanism of inhibition may be protein-protein interaction, which is the way in which HIV-1 protease inhibits the homologous retroviral reverse transcriptase (Ng et al., 1997).

In summary, a protein with potent antiproliferative activity toward tumor cells and inhibitory activity toward HIV-1 reverse transcriptase and some antifungal activity was isolated from C. spinosa seeds. The N-terminal sequence of the isolated protein bears some similarity to the partial sequence of imidazoleglycerol phosphate synthase but not to known proteins with antiproliferative, HIV-1 reverse transcriptase inhibitory or antifungal activities.

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Sze-Kwan Lam *, Tzi-Bun Ng

Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

* Corresponding author.

E-mail address: lamszekwan[A]yahoo.com.hk (S.-K. Lam).

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doi:10.1016/j.phymed.2008.09.006
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Title Annotation:human immunodeficiency virus
Author:Lam, Sze-Kwan; Ng, Tzi-Bun
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:May 1, 2009
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