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A mannose-binding lectin from Sophora flavescens induces apoptosis in HeLa cells.

Abstract

The objective of this study was to investigate the anti-tumor activity of a lectin from Sophora flavescens and explore its potential apoptotic induction mechanism. Here, an elegant series of biochemical and cell biology methods were carried out in a sequential procedure (e.g., MTT, cell morphologic changes and LDH assays, DNA ladder as well as flow cytometric assay). As a result, we found that this lectin shows a strong cytotoxicity against HeLa cells and induces apoptosis in a time- and dose-dependent manner. Subsequently, according to caspase inhibition and Western blot analysis, we further demonstrated that it is a typical caspase-dependent apoptotic mechanism. Furthermore, we also exerted some bioinformatics methods to identify the mannose-binding specificity of this lectin. In conclusion, all experimental results demonstrated that this lectin seems to be a potent anti-tumor agent for its cytotoxicity and apoptosis effects on HeLa cells. Also, bioinformatics analyses showed that this lectin is speculated to bind a certain mannose-containing receptor on cancer cell surface thereby initiating downstream caspase cascade.

[c] 2008 Elsevier GmbH. All rights reserved.

Keywords: Sophora flavescens lectin; Anti-tumor; Apoptosis; Caspase; Mannose-binding activity

Introduction

Plant lectins constitute a heterogeneous group of proteins with different biochemical properties and carbohydrate-binding specificities. They are widespread throughout the plant kingdom, occurring in a number of plant species from all major taxonomic groupings (Van Damme et al., 1998b). Advances in the structural analysis and molecular cloning of lectin genes enable subdivision of plant lectins in a limited number of subgroups of structurally and evolutionary related proteins. Four major lectin families, namely, the legume lectins, the chitin-binding lectins composed of hevein domains, the type2 ribosome-inactivating proteins and the monocot mannose-binding lectins comprise the majority of all currently known plant lectins (Van Damme et al., 1998a).

In the previous study, several lectins, present in beans and other edible plant products, have been shown to have markedly anti-tumor activities (Pryme et al., 1996; Ryder et al., 1998). A number of investigations have also indicated that the lectins isolated from mistletoe could induce apoptosis in a series of tumor cell lines (Bantel et al., 1999). To our knowledge, apoptosis is the programmed process utilized to eliminate redundant or potentially deleterious cells, and apoptosis induction is arguably the most potent defense against cancer, making it a desirable end point for cancer therapy (Hengartner, 2000; Meier et al., 2000). Thus, some lectins would be potent anti-cancer candidate agents if they possess remarkably apoptotic induction activities against a variety of cancer cell lines.

A mannose-binding lectin (designated SFL) was isolated from roots of Sophora flavescens Ait which has been used as a Chinese traditional medicine for thousands of years. The molecular weight of SFL was reported to be 32 kDa and it agglutinated both rabbit and human erythrocytes (Deng et al., 2000). Furthermore, the full-length cDNA coding SFL was cloned and sequenced (GenBank AF285121). The deduced amino acid sequence indicates that a pre-protein with 284 amino acid residues is firstly translated and then processed to a mature protein with 254 amino acids (Ma et al., 2001). Also, SFL is a member of legume lectins that is one of the most extensively studied families of plant lectins and is considered as a model system to study the molecular basis of protein-carbohydrate interactions for several decades. Previous studies have established that the deduced amino acid sequence of SFL consists of 254 amino acids and Asn182 was an N-glycosylation site (Ma et al., 2001). Importantly, SFL has been reported for its hemagglutinating activity and antifungal activity (Deng et al., 2000). However, whether SFL possesses a remarkable anti-tumor activity and its potential apoptosis induction mechanism still remains to be discovered.

In the present study, we reported that SFL caused HeLa cell death by both direct cytotoxicity and apoptosis. Interestingly, we found that the apoptosis induced by SFL is in a typical caspase-dependent manner. Moreover, bioinformatics methods demonstrated that SFL possesses the mannose-binding activity, which might be correlated to the apoptotic mechanisms based upon some previous studies that tumor cells express high levels of mannose branches on the membrane (Dennis et al., 1987). As mentioned above, these results would provide a crucial clue to seek the possible potential apoptotic mechanisms; also, they would provide new evidence for understanding more significant biological implications of SFL in impending cancer therapies in future investigations.

Materials and methods

Source of Sophora flavescens Ait and HeLa cell culture

Sophora flavescens Ait was purchased from Chengdu Chinese traditional medicine market. SFL was purified from the rhizomes of Sophora flavescens Ait as described by Deng et al. (2000). HeLa cells were provided by Medical Sciences Center of West China in Sichuan University. The cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 0.03% L-glutamine (Gibco) and maintained at 37 [degrees]C with 5% [CO.sub.2] in a humidified atmosphere.

MTT colorimetric assay

Tests were prepared as the method referred by Mosmann (1983). HeLa cells with logarithmic growth phase (1 x [10.sup.5] cells/ml) were seeded independently in a 96-well plate with the final volume 100 [micro]l containing 1 x [10.sup.4] cells per well. These plates were incubated at 37 [degrees]C for 24 h. And then various concentrations of SFL were added. After another 24h, 0.05 mg (10 [micro]l of 5 mg/ml) MTT was added to each well and incubated at 37 [degrees]C for 4h. The absorbance of the samples was measured at 570 nm with a spectrophotometer [Model 3550 Microplate Reader (Bio-Rad)]. The percentage of cell growth inhibition was calculated as follows:

Cell viability (%) = OD570 (drug)/OD570 (control) x 100.

Observations of cell morphologic changes and nuclear damage

HeLa cells in RPMI-1640 containing 10% FBS were seeded into 96-well culture plates and cultured for 24h. Hela cells were treated with the 0.05% dimethyl sulfoxide (DMSO). SFL (1 x [10.sup.-3] mg/ml) was added to the cells and the cellular morphology was observed using phase contrast microscopy (Leica, Wetzlar, Germany).

Apoptotic nuclear morphology was assessed using Hoechst 33258 (St. Louis, MO, USA, staining). The cells were fixed with 3.7% paraformaldelyde for 30 min at room temperature, and then washed and stained with 167 [micro]m Hoechst 33258 at 37 [degrees]C for 30 min. The cells were washed and suspended again respectively in PBS for morphologic observation under a fluorescent microscope (Olympus, Tokyo, Japan).

DNA fragmentation assay

HeLa cells cultured with or without SFL (1 x [10.sup.-3]mg/ml) at 37 [degrees]C for 12, 24 and 48h were harvested and suspended with 1 ml medium. The total DNA was isolated by using a DNA extraction kit (Shanghai Wason Co. Ltd.) and analyzed by electrophoresis on 1.5% agarose gel containing 0.1 [micro]g/ml ethidium bromide and visualized under UV light.

Lactate dehydrogenase (LDH) activity-based cytotoxicity assays

LDH activity was assessed using a standardized kinetic determination kit (Zhongsheng LDH kit, Beijing, China). LDH activity was measured in both floating dead cells and viable adherent cells. The floating cells were collected from culture medium by centrifugation (240g) at 4 [degrees]C for 5 min, and the LDH content from the pellets was used as an index of apoptotic cell death (LDHp) (Zhang et al., 2004). The LDH released in the culture supernatant (extracellular LDH, or LDHe) was used as an index of necrotic death, and the LDH present in the adherent viable cells as intracellular LDH (LDHi). The percentage of apoptotic and necrotic cell death was calculated as follows:

Apoptosis % = LDHp/(LDHp + LDHi + LDHe) x 100

Necrosis % = LDHe/(LDHp + LDHi + LDHe) x 100

Flow cytometric analysis

HeLa cells cultured with or without SFL (1 x [10.sup.-3] mg/ml) at 37[degrees]C for 12, 24 and 48 h were harvested, washed with 0.01 M cold phosphate-buffered saline (PBS) and fixed with 70% ethanol at 4[degrees]C for 24 h. Then cell pellets were suspended in 1 ml PI solution containing 65 [micro]g/ml Rnase and 0.1% (w/v) Triton X-100 in sodium citrate (3.8 mM), followed by incubation on ice in the dark condition for 1 h. Samples were analyzed by a FACS Flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Effects of caspases on SFL-induced cytotoxicity in HeLa cells

HeLa cells were treated with 1 x [10.sup.-3] mg/ml SFL for 12, 24, 36 and 48 h. Both adherent and floating cells were collected, and then Western blot analysis was carried out. Equal amounts of total protein were separated by electrophoresis in 12% SDS polyacrylamide gel electrophoresis and blotted onto a nitrocellulose membrane (Meuillet et al., 2000). Proteins were detected using polyclonal antibody and visualized using anti-rabbit, anti-mouse or anti-goat IgG conjugated with peroxidase (HRP) and 3, 3-diaminobenzidine tetrahydrochloride (DAB) as the HRP substrate. At the same time, caspase inhibitors were used to confirm involvement of caspases in SFL-induced cell death. The cells were incubated with z-DEVD-fmk (caspase-3 inhibitor, 20 [micro]mol/1), z-IETD-fmk (caspase-8 inhibitor, 20 [micro]mol/[1.sup.-1]), z- LEHD-fmk (caspase-9 inhibitor, 20 [micro]mol/1), z-AEVD-fmk (caspase-10 inhibitor, 20 [micro]mol/1) and z-VAD-fmk (pan-caspase inhibitor, 20 [micro]mol/1) for 1 h, and then treated with SFL for 24 h.

Statistical analysis of the data

All the data were confirmed in at least three independent experiments. These data were expressed as mean [+ or -] S.D. Statistical comparisons were made by Student's t-test. p < 0.05 was considered significantly.

Bioinformatics analyses

The lectin sequences from Maackia amurensis (NCBI Entry AAB39934), Ulex europaeus (NCBI Entry AF190633), Lens culinaris (NCBI Entry AAY21161), Vicia faba (NCBI Entry CAD27436), Arachis hypogaea (NCBI Entry AY431029) and Canavalia ensiformis (NCBI Entry AF308777) were acquired from the GenBank database. The alignment was running by using the program CLUSTALW (version 1.81) (Thompson et al., 1997).

The sequence of Sophora flavescens lectin (SFL) was also retrieved from the GenBank database (NCBI Entry AAG00508), Swiss-Model (Guex and Peitsch, 1997; Schwede et al., 2003) was utilized to build its three-dimensional structure, and docking experiments were carried out by using AutoDock (Morris et al., 1998).

Results

Cytotoxic effects of SFL on HeLa cells

SFL induced HeLa cell death in a dose-dependent manner. SFL from 0.01 x [10.sup.-3] to 5 x [10.sup.-3] mg/ml exerted a potent inhibitory effect on the growth of HeLa cells (Fig. 1). When HeLa cells were incubated with 1.0 x [10.sup.-3] mg/ml SFL after 24 h, the inhibitory ratio reached nearly 50%. Therefore, 24h incubation with 1.0 x [10.sup.-3] mg/ml SFL is sufficient for half inhibition of cell growth ([IC.sub.50]).

[FIGURE 1 OMITTED]

Observations of cellular configuration

When HeLa cells were cultured with SFL for 24 h, morphologic changes were confirmed by Hoechst 33258 staining. In the control group, the nuclei in which DNA resides were round and homogeneously stained (Fig. 2A), whereas the SFL-treated cells showed manifest fragmented DNA in nuclei (Fig. 2B). Consistent with the result above, marked apoptotic morphologic alterations including membrane blebbing and nuclear condensation were also observed by phase contrast microscopy (Fig. 2D). These suggested that SFL could induce Hela cells apoptosis.

[FIGURE 2 OMITTED]

DNA ladder assay of apoptosis in HeLa cells

The most distinctly biochemical hallmark of apoptosis is the activation of the endogenous [Ca.sup.2+]/[Mg.sup.2+] -dependent endonuclease and endonuclease-mediated cleavage of internucleosomes to generate oligonucleotide fragments with about 180-200 bp length or their polymers (Wyllie, 1980). Characteristic ladder bands can be obtained by agarose gel electrophoresis of DNA extracted from apoptotic cells. In this study, DNA ladder bands could be observed in the 1 x [10.sup.-3] mg/ml SFL-treated group after 24 h, whereas in the presence of SFL for 12 h or in control group, smear-like DNA degradation was observed (Fig. 3). These phenomena demonstrated that SFL induced HeLa apoptosis in a time-dependent manner.

[FIGURE 3 OMITTED]

The balance between apoptosis and necrosis

To further characterize SFL-induced HeLa cell death was accomplished by apoptosis or necrosis, the ratios of apoptosis and necrosis in cells were analyzed by LDH activity-based assay. In the presence of 1 x [10.sup.-3] mg/ml SFL, the number of apoptotic and necrotic cells was 40% and 23% at 24h, respectively, suggesting that necrosis was also triggered by SFL in HeLa cells, whereas the number of apoptotic cells was much higher than necrotic cells. With the progressively increasing concentration of SFL, more and more necrotic cells appear. So we can say that the major cause of SFL-induced HeLa cell death was not necrocytosis but apoptosis (Fig. 4).

[FIGURE 4 OMITTED]

Apoptosis analysis by FACS

Healthy HeLa cells exhibited normal cell cycle characteristics ([G.sub.1]/[G.sub.0] and [G.sub.2]/M phases): the percentage of cells in apoptotic phage was 2.3%, at the same time, 13.3% of the cells were in [G.sub.2]/M phase (Fig. 5A). After incubation with SFL for 12h, 5.5% of cells had undergone apoptosis and the percentage of cells in [G.sub.2]/M phase was reduced to 11.99% (Fig. 5B). However, after 24h, the apoptotic ratio increased to 17.3% while the cells in [G.sub.2]/M phase reduced to 7.48% (Fig. 5C). After incubation with SFL for another 24 h, 25.8% of HeLa cells had undergone apoptosis and the percentage of HeLa cells in [G.sub.2]/M phase reduced to 3.7% (Fig. 5D). These results indicated that with the time of SFL treatment extending, the cell cycle of HeLa cells significantly altered, that the apoptotic ratio gradually increases and the percentage of HeLa cells in [G.sub.2]/M phase sharply reduces, whereas the percentage of cells in [G.sub.1]/[G.sub.0] phase did not change accordingly. It suggested that SFL induces HeLa cells apoptosis.

[FIGURE 5 OMITTED]

Effects of caspases on SFL-induced cytotoxicity in HeLa cells

Caspases are a family of cysteine proteases whose activated during the apoptotic processes. Western Blot analyses was carried out to confirm the participation of caspase-8 and caspase-3. In this study, procaspase-8 began to be degraded after 12h, suggesting its activation. The activated caspase-8 is released into the cytoplasm where it functions as a caspase initiator, activating downstream executioner caspase, primarily the procaspase-3. The 32-kDa band of procaspase-3 was observed after treatment with SFL for 12h, indicating the activation of caspase-3 (Fig. 6).

[FIGURE 6 OMITTED]

To further evaluate the involvement of caspases in SFL-induced cell death, five caspase inhibitors, z-DEVD-fmk (caspase-3 inhibitor), z-IETD-fmk (caspase-8 inhibitor), z-LEHD-fmk (caspase-9 inhibitor), z-AEVD-fmk (caspase-10) and z-VAD-fmk (pan-caspase inhibitor) were applied. After 24h incubation with SFL, the inhibitors of caspase-3, -8, -9, -10 and pan-caspase had inhibitory effects on cell death (Fig. 7).

[FIGURE 7 OMITTED]

Multiple sequence alignment and loop analyses

The experiments above showed that the SFL has an effect to induce apoptosis on HeLa cells. This effect may associate with its mannose-binding activity as it is known that tumorigenesis and metastasis always accompany the expression of high mannose type oligosaccharides on the cancer cell surface (Dall'olio, 1996; Dennis et al., 1987; Laidler and Litynska, 1997; Orntoft and Wolf, 1998).

According to Sharma and Surolia (1997), the carbohydrate-binding site of all legume lectins consists of residues which belong to five polypeptide loops (A-E). Loops A and B contain the essential aspartate (D) and glycine (G) residue, respectively. These two loops do not show much discrepancy among different lectins. In the current sequences, this picture is confirmed. Loop C is the metal-binding loop and wraps around the structurally important calcium and manganese ions. In the known sequences, there are always five different loop sizes, ranging from 12 to 16 residues, while SFL has a length of 16 residues. In contrast to loops A-C, loop D is highly variable in length and sequence and is often referred as the monosaccharide specificity loop. In the SFL sequence, the loop of ten residues is identical with that found in other known sequences of Man/Glc-specific lectins. Finally, loop E is found to interact with mannose in only a few cases; the docking experiment in the following text show that this loop is not involved in carbohydrate binding in SFL (Fig. 8). Therefore, the results of the loop analyses indicated that SFL contains all of the carbohydrate-binding associated residues which are the base of the carbohydrate-binding activity.

[FIGURE 8 OMITTED]

Docking experiments analysis

To get a better understanding of the potent mechanism, we determine the mannose-binding model and the binding energy of SFL by docking experiments. The result showed that a few amino acid residues (Asp84, Glyl04, Asnl36, Gln220 and Gln221) belonging to the four loops located at the top of the dome-shaped lectin monomer forming a mannose-binding site are responsible for the mannose-specific recognition. Thus, the hydrogen bonds, which were required to anchor the mannose into the binding site, could be formed. These residues create a network of seven hydrogen bonds with O3, O4, O5 and O6 of the sugar (Fig. 9). The lowest binding energy and docking energy are -6.93 and -7.17 kcal/mol respectively.

[FIGURE 9 OMITTED]

Discussion

Legume lectins, which represent the largest and most thoroughly studied family of these sugar-binding proteins, have received much attention for their remarkable anti-tumor activities in potentially cancer therapeutical applications (Sharon and Lis, 2002). In the present study, we found that SFL possesses an obvious cytotoxicity against HeLa cells and induces apoptosis in a time-and dose-dependent manner. More importantly, a typical caspase-dependent apoptotic mechanism was discovered as well. Based upon all evidence reported above, SFL seems to be a potential anti-cancer candidate agent for its strong anti-tumor activity ([IC.sub.50] = 1 x [10.sup.-3]mg/ml, 24 h); however, the important upstream apoptotic mechanisms still remain unclear.

Taking advantage of bioinformatics approaches, we analyze and predict the possible apoptosis mechanisms of SFL. Previous studies have established that tumor cells express high levels of mannose branches (that is, -GlcNAc beta l-6Man alpha 1-6Man beta 1-) on their surface (Dennis et al., 1987). In light of the mannose-binding specificity of SFL, we reasonably speculated that SFL is able to bind the mannose branches on the cancer cell surface. Taking consideration of caspase inhibition assay and Western blot analysis, we drew a putative conclusion that the lectin after binding to the high mannose type oligosaccharides receptor on HeLa cell membrane would be internalized and accumulated preferentially into the death-inducing signaling complex. Of note, the death-receptor pathway is triggered by members of the death-receptor superfamily (Hengartner, 2000). The complex with this lectin recruits multiple procaspase-8 molecules, resulting in caspase-8 activation. Then the death-receptor pathway converges at the caspase-3 activation, which plays a key role as the central 'executioner' in the process of apoptosis (Gerl and Vaux, 2005). As mentioned above, these results would provide a crucial clue for a better understanding of these apoptotic mechanisms in further investigations.

Furthermore, a number of lectins have been widely studied in anti-cancer researches for several years. Con A, a widely studied legume lectin, is cytotoxic or inhibitory to numerous cancer cell lines, has been reported for its remarkable anti-tumor activity (Chang et al., 2007; Lei and Chang, 2007). Also, Con A has been found to be mediated by autophagy (another programmed cell death) in mitochondrial pathway but the typical caspase-dependent apoptosis was not observed (Chang et al., 2007). Regarding that Con A and SFL are both legume lectins, the SFL should have possessed the same or similar apoptotic mechanism with Con A; however, here, we found a typical caspase-dependent apoptosis pathway of SFL through caspase inhibition and Western blot analysis. The distinction of their antitumor mechanisms might be due to the fine differences of their carbohydrate-binding sites, even their tertiary structures. Furthermore, mistletoe lectin I, belonging to the ribosome-inactivating protein (RIP II) family was reported to exert potent immunomodulating and cytotoxic effects, and it triggered a receptor-independent mitochondria-controlled apoptotic pathway (Bantel et al., 1999). In contrast, SFL, a member of legume lectins, has a distinctive carbohydrate-binding site and three-dimensional structure with mistletoe lectin. Thus, the anti-tumor mechanisms of them should be different just as we speculated that the apoptotic induction of SFL should be dependent on death-receptor pathway. Accordingly, this interesting phenomenon would be a principal force to further study the relationship between the tertiary structure and biological activity in legume lectins.

Although bioinformatics methods are the best ways to tackle the putative mechanism, questions remain about the reliability of these predictions. More thoughtful analyses of biochemical and molecular experiments would better explain and ensure the extent and significance of this putative mechanism. In summary, the finding of SFL exhibiting a remarkable apoptosis-inducing activity in a typical caspase-dependent manner would open out a new exploration for natural lectins as anti-cancer compounds. Furthermore, it may, therefore, provide more promising insights into pharmaceutical exploitation in treatment of cancer diseases in future.

Acknowledgments

We are grateful to Dr. Xiang-jiu He for his significant discussions and access to more useful information. Subsequently, we also thank Dr. Yan Cheng for her skillful assistance with the manuscript. Furthermore, this work was supported by grants from the National Natural Science Foundation of China (General Programs: No. 30270331 and No. 30670469). Also, this work was supported by Director Fund of State Key Laboratory of Oral Diseases (Sichuan University); The Science and Technology Fund for Distinguished Young Scholars of Sichuan Province (No. 06ZQ026-035) and the Key Technologies R & D Program of Sichuan Province (2006Z08-010).

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* Corresponding author at: College of Life Sciences, Sichuan University, Chengdu 610064, China. Tel.: + 86 28 85410672; fax: + 86 28 85417281.

E-mail address: jinkubao@yahoo.com (J.-K. Bao).

(1) Co-first authors.

0944-7113/$-see front matter [c] 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2008.02.025

Zhen Liu (a), (1), Bo Liu (a), (1), Zi-Ting Zhang (a), Ting-Ting Zhou (b), He-Jiao Bian (a), Ming-Wei Min (a), Yan-Hong Liu (a), Jing Chen (a), Jin-Ku Bao (a), (c), *

(a) College of Life Sciences, Sichuan University, Chengdu 610064, China

(b) Shanghai Key Laboratory for Pharmaceutical Metabolite Research, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, China

(c) ''State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610064, China
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Author:Liu, Zhen; Liu, Bo; Zhang, Zi-Ting; Zhou, Ting-Ting; Bian, He-Jiao; Min, Ming-Wei; Liu, Yan-Hong; Ch
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Oct 1, 2008
Words:4353
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