Ardipusilloside I purified from Ardisia pusilla competitively binds VEGFR and induces apoptosis in NCI-H460 cells.
The present study was to evaluate the effects of Ardipusilloside I isolated from Ardisia pusilla on the growth, vascular endothelial growth factor receptor (VEGFR) expression and apoptosis of NCI-H460 cell line by MTT, ELISA and flow cytometer, respectively. The docking assay between Ardipusilloside I and VEGFR was studied by Sybyl/Sketch module. The change of microstructure was observed by transmission electron microscope (TEM). DNA fragmentation was visualized by agarose gel electrophoresis. The protein expression of Bax and Bcl-2 was detected by immunohistochemistry (1HC). A series of changes were observed in NCI-H460 cell treated by Ardipusilloside I, including microstructure, DNA fragmentation, protein expression of VEGFR, Bax and Bcl-2. The results showed Ardipusilloside I had a good docking with VEGFR and could inhibit growth and induce apoptosis of NCI-H460 cell in a dose-dependent manner. Cell cycle was significantly stopped at the [G.sub.1] phase. Under electronic microscope, the morphology of NCI-H460 cell treated with Ardipusilloside I showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body. VEGFR and Bcl-2 expression were decreased and Bax expression was increased. In conclusion, all these results demonstrate that Ardipusilloside 1 has a good docking with VEGFR and has an inhibitory effect on growth of NCI-H460 cell and can induce its apoptosis.
Ardisia pusilla A. DC (of the Myrsinaceae family), a Chinese medicinal herb also known as Jiu Jie Long (JJL), is the source of the triterpenoid saponins called ardipusillosides. In previous studies, Ardipusilloside I has shown anticancer activity in both Lewis pulmonary carcinoma and hepatocarcinoma (Tao et al. 2005a, b). However, there are few reports about its effect on apoptosis. It is currently unknown whether the antitumor effect of Ardipusilloside I is caused by binding VEGFR, reduced cell growth and increased apoptosis. So, we used NCI-H460 cell lines to study the effect of Ardipusilloside I at different concentrations on cell viability and apoptosis.
Apoptosis or programmed cell death is an active, genetically controlled process of cell suicide. Several biochemical features have been identified as associated to apoptotic cell death. Among these, cleavage of genomic DNA into multiple fragments is the most typical feature of the apoptotic process (Wyllie 1980). This characteristic ladder pattern genome fragmentation is well visualized by agarose gel electrophoresis of the isolated DNA (Wyllie et al. 1984). Apoptosis is an active cell death process, which requires specific protein and gene regulation. Bcl-2 family proteins are one of the already identified regulators of apoptosis. Bcl-2 family of homologous proteins represents a critical checkpoint within most apoptotic pathways, acting upstream of such irreversible damage to cellular constituents (Adams and Cory 1998). At least 15 Bcl-2 family members have been identified so far in mammalian cells. They function either as pro-apoptotic (Bax, Bak and Bad) or anti-apoptotic (Bcl-2 and Bcl-XL) regulators. These proteins form heterodimers of anti- and pro-apoptotic members, thereby titrating one another's function (Adams and Cory 1998). The ratio of anti- and pro-apoptotic proteins determines in part how cells respond to apoptotic or survival signals (Farrow and Brown 1996).
There was correlation between Bcl-2 and VEGFR. The over-expression of Bcl-2 was related to tumor angiogenesis (Nor et al. 2001). Bcl-2 could up-regulate VEGF level leading to tumor angiogenesis, whereas the VEGF could up-regulate Bcl-2 level inhibiting cell apoptosis (Pidgeon et al. 2001). VEGF is one of the most important inducers of angiogenesis and exerts its cellular effects by interacting with VEGFR. VEGFR is the major positive signal transducer for endothelial cell proliferation and differentiation. For these reasons, VEGFR appears to be an interesting target for design of anticancer agents (Gasparrini et al. 2005).
In this study, we found that Ardipusilloside I had a good docking with VEGFR. So, we investigated the inhibition on VEGFR and the effect on apotosis of Ardipusilloside I. The results showed Ardipusilloside I could competitively bind VEGFR and induce apoptosis of NCI-H460 cell, and inhibit its growth in a dose-dependent manner. The cell-cycle varied with increase of [G.sub.1] phase cells and decrease of S phase cells by Ardipusilloside I. There were karyopyknosis, chromatin agglutination and apoptosis bodies under transmission electron microscope (TEM) and DNA ladder by ultra-violet spectroscopy (UV) when NCI-H460 cells were exposed to Ardipusilloside I at different concentrations. The results of immunohistochemistry (IHC) showed that Ardipusilloside I could down-regulate Bcl-2 protein level and yet up-regulate Bax level.
Our results demonstrated that Ardipusilloside I selectively caused growth arrest and apoptosis in NCI-H460 cells and this appeared to be mediated by the regulation of Bcl-2, Bax and VEGFR. These findings indicate that the occurrence of accelerated apoptosis in NCI-H460 cells is an important event in the process of anti-angiogenesis. Thus apoptosis triggered by Ardipusilloside 1 may have important implications for pharmacological attempts at its anti-angiogenesis.
Materials and methods
JJL was collected in Sichuan, China. Identification of the plant was done at the Pharmacognosy Laboratory in the Department of Pharmacy, School of food and Pharmacy, Zhejiang Ocean University where a voucher specimen is deposited. DMSO, MTT, RPMI-1640 and trypsin were from Sigma (St. Louis, MO, USA). Propidium iodide (PI) and RNase were from Sigma. Fluorouracil (5-Fu) was from Nantong Jinghua Pharmaceutical Co. Ltd. DNA extraction kit was from Shanghai Wason Co. Ltd. (Shanghai, China). Annexin-[delta]FITC Kit was from Jingmei BioTech (Shenzhen, China). ELISA kit for human VEGFR was purchased from R&D Systems (Minneapolis, MN, USA). Polyclonal rabbit anti-Bcl-2 and anti-Bax were purchased from Santa Cruz Biotech (CA, USA.). Other reagents used were analytical grades. NCI-H460 cell was purchased from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences. NCI-H460 cell was grown in RPMI 1640 medium containing 10% bovine serum, antibiotics (100 IU/ml penicillin and 100 [micro]g/ml streptomycin), at 37 C in a 5% [CO.sub.2] atmosphere.
Extraction and isolation of Ardipusilloside I
Air-dried and powdered JJL was extracted with 95% EtOH for 2 h and 75% EtOH for the second time. The extract was concentrated under decompression. The extracta sicca was mixed with water. The mixture was allowed to stand to separate into two layers and filtered. The filtrate was extracted by petroleum ether, aether, acetoacetate and n-butanol in turns. The extracta sicca by n-butanol was mixed with acetone to collect the precipitate. The precipitate components were separated systematically by column chromatography (CC, Silica Gel). Ardipusilloside I was isolated using a mixture of [CHCI.sub.3]/MeOH/[H.sub.2]O (65:35:10 by vol.) as a mobile phase and further purified by crystallization. Ardipusilloside I isolated by this procedure was over 99% in purity, as determined by high-pressure liquid chromatography (HPLC). The chromatographic conditions were as follows: a Sinochchrom ODS-BP column (200 mm x 4.6 mm; I.D. 5 [micro]m) was used. The mobile phase was methanol/[H.sub.2]O (80:20, v/v) with a flow rate of 1.0 ml/min at a column temperature of 25[degrees]C, and the detection wavelength was set at 205 nm. Ardipusilloside I is a triterpenoid saponin and its chemical structure is shown in Fig. 1. Ardipusilloside I was dissolved in water and diluted to the desired concentrations immediately before use.
[FIGURE 1 OMITTED]
In an effort to elucidate the binding modes of Ardipusilloside I with VEGFR, it was constructed with Sybyl/Sketch module and optimized using Powell's method with the Tripos force field with convergence criterion set at 0.05 kcal/([Angstrom] mol), and assigned with Gasteiger-HUckel method (Mou et al. 2009). The docking study performed using Sybyl/FlexX module, the residues in a radius of 6.5 A around the VEGFR (PDB ID: 1VPP) were selected as the active site. Other docking parameters implied in the program were kept default.
Assay of cell viability/cell growth
The effect of Ardipusilloside I on NCI-H460 cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium broide (MTT) assay (Naruse et al. 2002). Briefly, exponentially growing cells were harvested and plated in 96-well plates at a concentration of 2 x [10.sup.4] cells/well. After 24 h incubation at 37[degrees]C, cells were treated with Ardipusilloside I at various concentrations (0, 1.00, 1.50, 3.00, 4.50, 6.00, 7.50, 9.00, 10.50, 12.00 and 13.5 [micro]g/ml) for 48 h. Then, 20 [micro]l of MTT (5 mg/ml) was added to each well and incubated at 37[degrees]C for 4 h. After the supernatant was discarded, 150 [micro]1 of DMSO was added to each well, and the optical density of cells was determined with a microplate reader (Bio-RAD instruments, USA) at 490 nm and expressed as absorbance values.
VEGFR secretion in vitro
NCI-H460 cells (5 x [10.sup.4] cells per well) were cultured in 24-well culture plates for 24 h. Then, the cells were incubated for another 24 h after the medium was changed to serum-free. When Ardipusilloside I was added to the well, the final concentrations for NCI-H460 cells were 1.35, 2.70, 5.40 [micro]g/ml. The 24-h cultured medium was collected. VEGFR protein concentrations were quantified by a commercially available VEGFR-ELISA kit (Shima-mura et al. 2001). ODs were measured at 490 nm.
Transmission electron microscopy
NCI-H460 cells were seeded at the concentration of 1 x [10.sup.5] cells in culture flask and incubated for 24 h. Then, the cells were incubated for another 24 h after the medium was changed to serum-free. The cells were exposed to different concentrations of Ardipusilloside 1 (0, 1.35, 2.70, 5.40 [micro]g/ml) for 24 h. The 24-h cells were harvested and fixed with 2.5% phosphate-buffered glutaraldehyde at 4[degrees]C overnight. The fixed cells were washed by phosphate buffer, post-fixed in 1% phosphate-buffered Os[O.sub.4], dehydrated in serial alcohol solutions, and embedded in embedding medium. Ultrathin sections (50 nm) were stained with uranyl acetate followed by lead citrate, and cell morphology was observed by TEM (HITACHI H-600, Japan) (Oh et al. 1997; Yang et al. 2005).
DNA fragmentation assay
NCI-H460 cells (1 x [10.sup.6] cells) cultured with (1.35, 2.70, 5.40 [micro]g/ml) or without Ardipusilloside I at 37[degrees]C for 48 h were harvested and suspended with 1 ml medium. According to the manufacturer's protocol, the total DNA was isolated by using a DNA extraction kit (Shanghai Wason Co. Ltd.) and analyzed by electrophoresis for DNA ladder formation on 1.5% agarose gel containing 0.1 mg/ml ethidium bromide and visualized under UV light (Hao et al. 2004; Oberhammer et al. 1993).
Cell cycle analysis
The NCI-H460 cells were cultured in 6-well culture plates and incubated for 24 h with Ardipusilloside I at concentrations of 0, 1.35, 2.70, 5.40 [micro]g/ml. The cell cycle was assessed according to the percentage of cells with DNA using the PI staining technique as described previously (Wang et al. 1997; Xiang et al. 2006). Briefly, after centrifugation, NCI-H460 cells (2 x [10.sup.6] cells) were gently resuspended in 1 ml 75% icecold alcohol solution and incubated in the dark at 4[degrees]C overnight. The fixed cells were washed by phosphate buffered solution (PBS) for three times. After centrifugation, they were incubated with 1 ml RNase of 50 [micro]g/ml and 1 ml PI of 60 [micro]g/ml in the dark for 30 min before analyzed one by one with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The obtained data were analyzed with the software of Modfit L.M.
NCI-H460 cells were seeded at the concentration of 1 x 105 cells in culture flask and incubated for 24 h. Then, the medium was changed to serum-free for 24 h. The cells were exposed to different concentrations of Ardipusilloside I (0, 1.35, 2.70, 5.40 [micro]g/ml) for 24 h, 5-FU 15 [micro]g/ml as the positive group. The 24-h cells were harvested, washed by PBS, and resuspended in binding buffer. 100 [micro]l [10.sup.5] cells were incubated with Annexin-V-FITC and PI in the dark for 15 min. The analysis was performed with the FACScan flow cytometer (Yang et al. 2005; Metrailler-Ruchonnet et al. 2007; Sun et al. 2007).
Bax and bcl-2 protein expressions
NCI-H460 cells were exposed to different concentrations of Ardipusilloside I (0, 1.35, 2.70, 5.40 [micro]g/ml) for 24 h, 5-FU 15 [micro]g/ml as the positive group. The 24-h cells were harvested and washed for 1HC. Immunohistochemical staining was performed on cell sections. After blocking endogenous peroxidases and unspecific binding, incubations were carried out using polyclonal rabbit anti-Bax-antibody and anti-Bcl-2-antibody at 4 [degrees]C, and further enhanced by biotinylated secondary antibodies and streptavidin-horseradish peroxidase conjugate. Slides were stained with DAB-chromogen and counterstained with haematoxylin (Hoffmann et al. 2007). To evaluate the staining of Bax and Bcl-2, optical density of positive cells whose cytoplasm was stained was analyzed with the quantity one[R], 1-D analysis software (Version 4.4, BioBad).
Data were expressed as mean [+ or -] S.D. Statistical analysis was performed using the statistical software SPSS10.0. ANOVA was used to analyze statistical differences between groups under different conditions, p < 0.05 was considered as significant.
Docking of Ardipusilloside I in the active site of VEGFR showed many H-bond interactions between oxygen of the inhibitor and amino acid residues of the enzyme. Virtual docking of VEGFR in complex with Ardipusilloside I also showed that Ardipusilloside I bound to the ATP binding pocket of the kinase domain (Fig. 2). From Fig. 2A, we can see that glycosyl group extends to one hole of the active site. Fig. 2B indicates the hydrogen bond density on the surface of the enzyme. Fig. 2C shows the interaction between the compound and the active domains in the ribbon structure representation.
[FIGURE 2 OMITTED]
Effect of Ardipusilloside I on NCI-H460 cell growth
We examined the effect of Ardipusilloside I on the growth of NCI-H460 cell by MTT. As shown in Fig. 3, Ardipusilloside I showed dose-dependent inhibition on cell growth. The 50% cell growth inhibition (IC50) of Ardipusilloside I was 5.92 [micro]g/ml. The cell growth was not affected significantly at concentrations ranging from 0.00 to 3.00 [micro]g/ml.
[FIGURE 3 OMITTED]
Effect on VEGFR secretion
ELISA for VEGFR showed that Ardipusilloside 1 could competitively bind VEGFR in a dose-dependent manner at low concentrations (1.35-5.40 [micro]g/ml) compared with the control group in NCI-H460 cell (p < 0.05). The VEGFR expression reduced obviously at different concentrations (Fig. 4). There were significant differences between the Ardipusilloside I group and the control group.
[FIGURE 4 OMITTED]
Morphological studies of apoptotic cell
After treatment with Ardipusilloside I, there was an increase in the number of unattached cells in all experiments. Under inverted microscope, these cells were rounded and had condensed or fragmented nuclei, suggesting apoptosis. Some cells were detached and fragmented into apoptotic bodies. Agarose gel electrophoresis in the DNA fragmentation assay showed the characteristic apoptotic DNA ladder. The DNA ladder is shown in Fig. 5A. Such a pattern corresponded to inter nucleosomal cleavage, which was characteristic of apoptosis. Control cells did not exhibit such DNA fragmentation. The morphological changes could be observed by TEM. In the control group, as can be seen from Fig. 5B, the morphous of NCI-H460 cells is normal. After exposure to different concentrations of Ardipusilloside I, NCI-H460 cells showed typical apoptosis morphology characterized by volume reduction, endochylema concentration, nuclear chromatin of maldistribution and nuclear fragmentation by TEM. The apoptosis bodies were observed in Fig. 5C with 5.40 [micro]g/ml Ardipusilloside I.
[FIGURE 5 OMITTED]
Effect of Ardipusilloside I on the NCI-H460 cell cycle
Obvious changes in cell-cycle distribution of NCI-H460 cells treated with Ardipusilloside I were characterized by increase of [G.sub.1] phase cells and decrease of S phase cells, suggesting that Ardipusilloside I led to accumulation of NCI-H460 cells at the [G.sub.1] phase (Fig. 6). The [G.sub.1] phase cells were 61.00% in the control group (Fig. 6A). After treatment with Ardipusilloside I (1.35, 2.70, 5.40 [micro]g/ml) for 24 h, the corresponding [G.sub.1] phase cells were 70.05% (Fig. 6B), 71.77% (Fig. 6C), 86.25% (Fig. 6D), respectively.
[FIGURE 6 OMITTED]
Evaluation of Ardipusilloside I-induced apoptosis in NCI-H460 cells by FACS analysis
Apoptotic NCI-H460 cells were measured by a fluorescence activated cell sorter. Annexin-V staining combined with PI staining was performed in cells in the control group and cells treated with Ardipusilloside I at different concentrations, and analyzed by flow cytometry. Early apoptosis was in right quadrant of a dot-plot graph using Annexin-V-FITC versus PI (Fig. 7). The apoptotic cells of the negative control group are shown in Fig. 7A. The apoptotic cells of the group treated with 5-Fu were 29.26% (Fig. 7B). Similar activity on NCI-H460 cells was observed in the group treated with Ardipusilloside I. After treatment with Ardipusilloside I (1.35, 2.70, 5.40 [micro]g/ml) for 24 h, the corresponding apoptotic cells were 11.62% (Fig. 7C), 19.91% (Fig. 7D), 39.68% (Fig. 7E), respectively.
[FIGURE 7 OMITTED]
Expressions of Bcl-2 and Bax proteins
In this analysis, we examined the expression level of Bax and Bcl-2 of cell apoptosis molecules. Cells were prepared with different concentrations of Ardipusilloside I (0, 1.35, 2.70, 5.40 [micro]g/ml) and used for IHC. Ardipusilloside 1 had obvious effects on the level of Bax and Bcl-2 proteins in NCI-H460 cells. Bcl-2 protein expression was reduced in Ardipusilloside I-treated cells, while Bax protein expression was increased with the concentrations of Ardipusilloside I rising from 1.35 to 5.40 [micro]g/ml. The results indicated that Ardipusilloside I down-regulated Bcl-2 protein level and up-regulated Bax level in NCI-H460 cells. There were significant differences in protein expressions between the Ardipusilloside I group and the control group (Table 1).
Table 1 Effect of Ardipusilloside I on Bcl-2 and Bax proteins expressions. Bcl-2 score (%) Bax score (%) Control group 80.28 [+ or -] 11.02 5.12 [+ or -] 1.36 5-FU group 25.28 [+ or -] 3.06 ** 52.38 [+ or -] 6.02 ** Ardipusilloside I-treated group 1.35 [micro]g/mL 60.85 [+ or -] 6.56 * 29.82 [+ or -] 1.98 ** 2.70 [micro]g/mL 31.14 [+ or -] 4.48 ** 42.44 [+ or -] 6.92 ** 5.40 [micro]g/mL 19.29 [+ or -] 1.78 ** 59.89 [+ or -] 8.02 ** Values are expressed as means [+ or -] SE (n = 10). * P < 0.05, ** P < 0.01, vs. control.
Ardipusilloside I was a component that had pharmacologic actions. The past work demonstrated that Ardipusilloside I had excellent inhibitory effects on tumor and improved the immunologic function (Tao et al. 2005a, b). We screened Ardipusilloside I from JJL. In this study, we investigated the action of competitive binding VEGFR and cell apoptosis induced by Ardipusilloside I.
This study showed that Ardipusilloside I had a good docking with VEGFR, could competitively bind VEGFR, obviously inhibit growth of NCI-H460 cell, and change the cell cycle. Proliferating cells arrested in any phase of the cell cycle eventually die by apoptosis (Romanova et al. 1996). [G.sub.1] phase was a key point in cell periodic duty and the target of drug action (Ormerod et al. 1994). The cell cycle assay indicated the S phase cells decreased and [G.sub.1] phase cells increased in the treated group compared with the untreated group. It implies Ardipusilloside I results in [G.sub.1]-arrest. The morphological studies indicated that NCI-H460 cell showed unobvious chromatic agglutination, slight chromatin margination and typical apoptotic body in turns with the increased concentrations of Ardipusilloside I. In addition, a characteristic apoptotic DNA ladder appeared by agarose gel electrophoresis. In the Annexin V/PI assay, Ardipusilloside I could induce NCI-H460 cell apoptosis in a dose-dependent manner. These data demonstrate that Ardipusilloside 1 could induce NCI-H460 cell apoptosis.
Bcl-2 is an anti-apoptotic regulator and Bax is a pro-apoptotic regulator (Metrailler-Ruchonnet et al. 2007). Bcl-2 and Bax could be expressed in harmony, which plays an important role in sustaining morph and function of NCI-H460 cells. The cells are active when Bcl-2 is overexpressed. On the contrary, cells are dead if Bax is overexpressed (Aikawa et al. 1997; Bernecker et al. 2003). The ratio of anti- and pro-apoptotic proteins determines in part how cells respond to apoptotic or survival signals (Farrow and Brown 1996). There is an intimate relation between apoptosis and angiogenesis. The ability of angiogenesis is weakened when the cell undergoes apoptosis (Tong et al. 2004; Starzec et al. 2006; Chen et al. 2004). Cell factors perform important functions in the process of cell apoptosis, for instance, vascular endothelial growth factor (VEGF) could inhibit cell apoptosis (Nor et al. 2001). The crucial regulators of the angiogenesis process associated with tumor development and metastasis are VEGF and VEGFR (Brych-tova et al. 2006). The IHC and ELISA results showed that Ardipusilloside I competitively bound VEGFR and inhibited bcl-2 protein level, and improved bax protein expression, which indicated that VEGFR, Bcl-2 and Bax participated in the process of HUVEC apoptosis induced by Ardipusilloside I.
In summary, Ardipusilloside I competitively binds VEGFR and inhibits NCI-H460 cell growth, and induces NCI-H460 cell apoptosis. Although its precise mechanism of action has not yet been thoroughly elucidated, the data from the present study demonstrate that Ardipusilloside I is capable of up-regulating Bax/Bcl-2 in NCI-H460 cell. This shows that Ardipusilloside I may become a cell apoptosis inducer, which presents a pathway to investigate its anti-angiogenesis mechanism.
This work was supported by National Natural Science Foundation of China (Grant No. 20502035). At the same time, the authors gratefully acknowledge Prof. Wenfang Xu and Huawei Zhu for the docking study.
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* Corresponding author. Tel.: +86 29 84776189;
E-mail address: email@example.com (X. Wang).
Yanmin Zhang (a), Youle Qu (b), Jie Zhang (a), Xiaojuan Wang (c), *
(a) School of Medicine, Xi'an Jiaotong University, Xi'an 710061, PR China
(b) School of Food and Pharmcy, Zhejiang Ocean University, Zhoushan 316004, PR China
(c) Department of Pharmaceutical Preparation, School of Stomatology, Fourth Military Medical University, Xi'an 710032, PR China
[C]2009 Elsevier GmbH. All rights reserved.
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|Title Annotation:||vascular endothelial growth factor receptor|
|Author:||Zhang, Yanmin; Qu, Youle; Zhang, Jie; Wang, Xiaojuan|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jun 1, 2010|
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