Antiproliferative effects of n-butyl-β-D-fructofuranoside from Kangaisan on Bel-7402 cells.

AIMS: Kangaisan is a powdered compound prescription of Traditional Chinese Medicine which has been used in cancers for many years in Hubei province, China. The purpose of this study was to investigate the antitumor effects of Kangaisan and screen bioactive components.
MATERIALS AND METHODS: 3-(4,5-Dimethythiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, DNA (Deoxyribonucleic acid) fragmentation assay, Western blot, and real time-polymerase chain reaction were used to investigate the antiproliferation effect of n-butyl-β-D-fructofuranoside on Bel-7402 cells. STATISTICAL ANALYSIS: All experiments were performed in triplicate and the results were expressed as mean ± standard deviation. Statistical analysis was performed with analysis of variance using Origin 8.0 software.
RESULTS: It was illustrated that treatment of Bel-7402 cells with various concentrations of n-butyl-β-D-fructofuranoside resulted in growth inhibition in both a dose-dependent and time-dependent manner. The arrest of G₀/G₁ phase was also induced (P < 0.05). The increasing of sub-G₁ cell population indicated the apoplectic characteristic (P < 0.05). Furthermore, the emerging of DNA fragmentation and the increase of Bax/Bcl-2 ratio and p53 expression suggested the possible mitochondrial apoptotic pathway (P < 0.05).
CONCLUSIONS: The results illustrate that Kangaisan showed anticancer effects and n-butyl-β-D-fructofuranoside extracted from Kangaisan can suppress Bel-7402 cells via interfering cell cycle and by inducing apoptosis.

Introduction

Medicinal plants are extensively used and natural products are considered to be excellent sources for new drug.[1] A variety of traditional medicines are being widely investigated to analyze their potential as therapeutic agents.[2] Traditional Chinese medicine (TCM) has held an important role in health care in China because of its 5000-year-old tradition. The Chinese system of medicine has identified hundreds of individual herbal extracts as well as several drug preparations, which claim to treat and/or prevent several diseases.[3] Some well-known TCM, such as Rhubarb, also known as Qinghaosu, which showed anticancer activities in mouse tumor models, set us good examples in cancer therapy using TCM.[4] Composite and complex TCM remedies, or compound prescription, which is about the use of complex mixtures of medicinal plants in contrast to single natural products, may act in a synergistic fashion to increase therapeutic effects.[5] However, due to the difficulty to identify and prove the synergistic or antagonistic effects of chemical constituents in herbal composite prescription, research in the healing mechanism of such type of TCM continues.

Kangaisan, a mixture of six kinds of herbal medicines, has been used in clinic for cancer therapy for many years. It was reported that Kangaisan had antitumor effects especially on hepatocellular cancer and cervical cancer. Gastric cancer, lung cancer are common malignancies in China. Hence Bel-7402, HeLa, HC, and A549 cells were chosen as the tumor cells while the Bel-7402 was chosen for further study on mechanism. Polygonum orientale L., also known as “Kiss-Me-Over-The-Garden-Gate,” occurs in China to Japan and southward to Australia and is usually found in open, wet places along streams at low and medium altitudes. Several investigations have reported that Polygonum orientale L. or its organic extracts have antiproliferative activity in human cancer cell lines, including HepG2, MGC, Hela, and Hce-8693. Hedyotis diffusa Willd., Paris polyphylla Sm., Panax quinquefolius Linn., Bupleurum rotundifolium L. and Panax pseudoginseng Wall. var. notoginseng (Burkill) Hoo et Tseng, the remaining five components of Kangaisan are also the best-known Chinese herbal medicines which have long been known for their therapeutic properties in terms of clearing away heat and toxic materials, promoting blood circulation as well as reducing swelling and pain.

The mechanism of the anticancer activity of Kangaisan has not been studied. This paper intends to evaluate the antiproliferation effects of the organic extracts on three human cancer lines and to isolate the monomeric compound from the most activated organic phase and determine the possible mechanism of cytotoxic effect associated with apoptosis on human Bel-7402 cell lines, which could be useful for identifying molecular targets for pharmacological treatment of hepatocellular cancer. It could help us get a clear perspective of the anticancer effect of Kangaisan.

Materials and Methods

Chemicals and Reagents

The herbal medicine compound Kangaisan powder was provided by The NO. 11 Hospital of Wuhan (code: Z20082911). Cell isolation and culture reagents were obtained from Invitrogen life Technologies (Lidingo, Sweden). Mouse antihuman β-actin, mouse anti-human Bcl-2, mouse antihuman Bax, and mouse antihuman p53 antibodies were purchased from DAKO (USA). Horse radish peroxidase conjugated goat antimouse secondary antibodies were from Pierce, and ECL plus Western Blotting Detection Reagents were from Amersham Biosciences UK Limited (Chalfont, St. Giles, Buckinghamshire, UK). All other chemicals and reagents were obtained from Sigma (St. Louis, MO, USA).

Preparation of Organic Extracts

As represented in the flow chart [Figure 1], the essential oil and polysaccharides form Kangaisan powers could be firstly extracted and then each organic phase was obtained by using Soxhlet apparatus. The organic extracts were later evaporated under reduced pressure to obtain the residue, using a rotary evaporator to dryness in order to obtain the respective lyophilized powder.

Figure 1

Extraction and partitioning of bioactive parts of the compound Fructus Polygoni Orientalis

Cell Culture

HGC, A549, HeLa, and Bel-7402 cell lines were provided by the Institute of Cell Biology (Shanghai, China). Bel-7402 cells were grown in DMEM (Dulbecco's Modified Eagle Medium), while others were in RPMI (Roswell Park Memorial Institute medium) 1640 medium. All of these were supplemented with 10% fetal calf serum, 100 U/mL of penicillin and 100 μg/mL of streptomycin, incubating at 37°C in a humidified air containing 5% CO2 and maintained as previously described.[6]

MTT Assay

MTT assay was used to determine cellular mitochondrial dehydrogenase activity reflecting initial cell death. In brief, the cells were plated in 100 μL media in 96-well plates at an initial density of 1 × 104 cells per well. On the following day, the experimental medium containing different concentrations of extracts were added and then incubated for varying times. A total of 50 μl of MTT solution (2 mg/mL in PBS (Phosphate buffered saline) ) was added to each well and incubated for 4 h. After careful removal of the media, 150 μL DMSO (Dimethyl sulfoxide) per well was added for further 30 min. Plates were read on an ELISA (Enzyme-linked immunosorbent assay) microplate reader (Molecular Devices, Sunnyvale, USA) at a wavelength of 560 nm and a reference wavelength of 620 nm. Cell viability was determined as the relative reduction of the optical density which correlates with the amount of viable cells in relation to cell control.[7]

Activity screening of organic extracts on tumor cell lines

MTT assay was used to detect the antiproliferation activities of each organic extracts on HGC, A549, and HeLa cell lines.

Extraction and Isolation

Lyophilized powder EtOH extract was chromatographed over silica gel (200~300 mesh) column eluted with a gradient of CHCl3 and MeOH (95:5) to yield three fractions (A 5.5 g, B 38 g, C 40 g). Fraction B was rechromatographed on a silica gel column (100~200 mesh, CHCl3-MeOH, 95:5) to yield a monomeric compound n-butyl-β-D-fructofuranoside (21 mg, C10 H20 O6 : Amorphous powder. [α]D + 96 ° (MeOH). IR (KBr): v/cm = 3446, 3359, 3286, 2989, 2956, 2929, 2887, 2744, 2684, 1454, 1375, 1334, 1261, 1190, 1122, 1032, 914, 768, 594, 555. 1H-NMR: DMSO-d6, 200MHz, 25C: δ/ppm = 2.88-3.75 (t,2H,J = 3.56Hz,H-7), 2.88-3.75 (m, 2H, J = 3.52Hz, H-4, 2), 2.88-3.75 (m, 5H, J = 8.43Hz, H-3, 5, 6), 1.46 (m, 2H, J = 3.67Hz, H-8),1.35 (m, 2H, J = 3.45Hz, H-9), 0.88 (t, 3H, J = 5.25Hz, H-10). 13C-NMR: 100.1 (C-1), 69.25 (C-2), 68.85 (C-3), 69.35 (C-4), 62.20 (C-5), 63.52 (C-6), 59.25 (C-7), 31.26 (C-8), 18.54 (C-9), 12.99 (C-10). FAB-MS: m/z = 236 (M+,259-Na), 154 (131+Na). EI-MS:m/z194 (M+-31), 149, 103, 85, 77,73, 60, 57, 43, 41. Its IR spectrum showed the characteristic absorption of hydroxyl groups (3510-3160 cm-1).), which was further indentified by IR (Infra red) spectra (WQF-200 Fourier transform infrared spectrometer), FAB-MS (Fast atom bombardment mass spectrometry) and EI-MS (ZAB-3F Electron ionization mass spectrometry) as well as 1H NMR and 13C NMR (Varian Merwry-vx 300 Nuclear Magnetic Resonance Spectroscopy).

Antiproliferation effect of n-butyl-β-D-fructofuranoside on Bel-7402 cells

Cytotoxicity of N-Butyl-β-D-Fructofuranoside on Bel-7402 Cells

Bel-7402 were seeded at 1 × 104 cells/well in 96-well plates and exposed to various concentrations (0.47, 2.95, 5.90, 11.8, 17.7, and 23.6 μg/mL). After incubation for 24, 48, and 72 h, viabilities were evaluated using the MTT assay.

Flow Cytometry Assay

Flow cytometry was used to quantitatively detect the apoptosis rate. After being treated with n-butyl-β-D-fructofuranoside at various concentrations (0, 50, and 75 μg/mL) for 48 h, cells were washed with PBS (pH 7.4) and fixed in 70% ethanol at 4°C overnight. After fixation, the cells were stained with PI at 1 mg/mL for 15 min at room temperature, determined by FACScan flow cytometry (Beckman, USA) and analyzed by CellQuest software (Beckman, USA).

Analysis of DNA Fragmentation

Cells were incubated with n-butyl-β-D-fructofuranoside 25, 50, and 75 μg/mL for 75 h and then analyzed by electrophoresis as previously performed. The DNA was extracted, fractionated in a 1.8% agarose gel, and visualized using ultraviolet fluorescence.

Western Blot Analysis of Bcl-2, Bax and p53

Bcl-2, Bax, and p53 from the Bel-7402 cells were detected by Western blotting. Bel-7402 cells were treated with the highest concentration (23.6 μg/mL) of n-butyl-β-D-fructofuranoside. At 24, 48, and 72 h, cells were harvested, washed twice with phosphate buffered saline and treated with a lysis buffer [1% Triton X-100, 0.15 M NaCl and 10 mM Tris (pH 7.4)] containing protease inhibitors (50 mg/mL phenylmethylsulphonyl fluoride, 2 mg/ml aprotinin and 2 mg/ml leupeptin) at 4°C for 30 min. The protein concentration was determined by the Bradford method. Equal amounts of total protein (60 μg) were separated on a 10% Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). After transfer onto a nitrocellulose membrane, protein loading was checked by Ponceau S staining and nonspecific binding was blocked with 1% BSA (Bovine serum albumin) in Tris-buffered saline-Tween 20 [0.15 M NaCl, 10 mM Tris (pH 8.0), 0.05% Tween 20] at 4°C overnight. Blots were probed with mouse anti-human Bcl-2 (1:400, DAKO, USA), mouse antihuman Bax (1:1000, DAKO, USA), and mouse antihuman p53 (1:1000, DAKO, USA) monoclonal antibodies. After incubation with horseradish peroxidase-conjugated antimouse immunoglobulin G, bands were visualized by chemiluminescent detection (ECLTM Western blotting analysis system, Amersham Italia Srl), following the supplier's recommended procedures. Prestained molecular weight markers were used as reference.

Quantitative Real-Time-PCR

The total RNA was extracted by RNeasy Mini Kit (Sigma, USA). Quantitation was done using a fluorescence based real-time detection method Rotor-gene 6000 Sequence Detection System (Sigma, USA). The polymerase chain reaction reaction mixture consisted of 600 nmol/L of each primer, 200 nmol/L probe, 2.5 U Taq polymerase, 100 μmol/L each dATP, dCTP, dGTP, 200 μmol/L dUTP, 2.5 mmol/L MgCl2, and 1 × TaqMan Buffer A containing a reference dye, to a final volume of 20 μL (all reagents Applied Biosystems). The cycle parameters used in this system were as follows: reverse transcription at 50°C for 10 min, initial activation at 94°C for 5 min, 40 cycles of denaturation for 20 sec at 92°C, 20 sec at 55°C, 20 sec at 72°C for annealing, and extension at 72°C. For each sample, parallel TaqMan PCR reactions were performed for each gene of interest and the β-actin reference gene to normalize for input cDNA (Complementary DNA). The obtained ratio between the values provides relative gene expression levels for the gene locus investigated.

Statistical Analysis

All experiments were performed in triplicate and the results are expressed as mean ± standard deviation. Statistical analysis was performed with analysis of variance using Origin 8.0 software.

Results

Six organic phases [Figure 1], BuOH extract, EtOH extract, EtOAc extract, MSO extract, Polysaccharides extract and essential oil extract were obtained from the compound Kangaisan [Figure 1].

The MTT test [Figure 2] showed that EtOH extract significantly inhibited the growth of HGC (Human gastric cancer cells), A549 and HeLa cell lines (P < 0.01) [Figure 2]. The viability of Bel-7402 cells was tested with different concentrations of n-butyl-β-D-fructofuranoside by MTT method, the results showed that n-butyl-β-D-fructofuranoside significantly inhibited the proliferation of Bel-7402 cells at both time- and dose-dependent manner [Figure 3], [Table 1]. The cell population of each phase was counted by flow cytometry [Figure 4]. The increase of G0/G1 phase and decrease of S and G2/M phase cells suggested that n-butyl-β-D-fructofuranoside could suppress Bel-7402 cell proliferation associated with cell cycle arrest in G0/G1 phase (P < 0.05) [Figure 4]. DNA isolated from Bel-7402 cells cultured with different concentrations of n-butyl-β-D-fructofuranoside showed a characteristic ladder pattern of apoptosis [Figure 5]. As shown in [Figure 6], n-butyl-β-D-fructofuranoside significantly decreased Bcl-2 and increased Bax and p53 levels in a time manner. The downregulation of Bcl-2 and upregulation of Bax and p53 by n-butyl-β-D-fructofuranoside exposure appeared after 24 h of drugs treatment (P < 0.05) [Figure 6].

Figure 2

Effect of organic extracts on the proliferation of three cell lines. Six bioactive parts of the compound Fructus Polygoni Orientalis were obtained. And the cell lines were treated with the extracts of 50μg/ml for 24 hours and the viabilities were assessed by MTT assay. The low OD values represent the low viability. Data are presented as means ± SD (n= 3). The asterisk indicates a significant difference between control and drug-treated cells (*P < 0.05, **P < 0.01)

Figure 3

Survival inhibition of hepatocellular carcinoma Bel-7402 cells by EtOH extract n-butyl-β-D-fructofuranoside assessed via MTT proliferation assay. (a) Cells were treated with n-butyl-β-D-fructofuranoside at concentrations 0.47, 2.95, 5.90, 11.8, 17.7 and 23.6μg/ml for 24, 48 and 72 hours. Nontreated and 0.1% DMSO-treated cells were used as control. n-butyl-β-D-fructofuranoside suppressed proliferation of Bel-7402 cells in both time- and dose-dependent manner. Each value represents means ± SD in three different experiments. The IC50 could be calculated via logistic curve fitting of cell viabilities for 24 (b), 48 (c) and 72 hours (d)

Table 1

Cytotoxic activities of n-butyl-β-D-fructofuranoside on Bel-7402 cells

Figure 4

Effect of n-butyl-β-D-fructofuranoside on cell cycle distribution in Bel-7402 cells. (a) After Bel-7402 cells were treated with n-butyl-β-D-fructofuranoside (0, 50 and 75μg/ml) for 48 hours, the cells were stained with propidium iodide, and the DNA contents were analyzed using flow cytometry. The 2N (diploid) and 4N (tetraploid) DNA contents represent the G1and G2/M phases, respectively, of the cell cycle. M1 indicate the percentage of apoptotic sub-G1 cells with hypodiploid DNA content. (b) The percentage of cells in different phases of cell cycle was represented by bar chart. Data are presented as means ± SD (n= 3). The asterisk indicates a significant difference between control and drug-treated cells (*P < 0.05)

Figure 5

DNA fragmentation. After HeLa cells were treated with various concentrations (25, 50 and 75μg/ml) of n-butyl-β-Dfructofuranoside for 48 h, the genomic DNA was extracted. Cellular fragmented DNA was analyzed by 1.8 % agarose gel electrophoresis. Lane M, DNA marker; lane 1, control; lane 2, 25μg/ml; lane 3, 50μg/ml; lane 4, 75μg/ml

Figure 6

Effect of n-butyl-β-D-fructofuranoside on the expression of three apoptosis-related proteins Bcl-2 (a), Bax (b) and p53 (c). Equal amounts of protein (130 μm) from the Bel-7402 cells treated with 23.6μg/ml drugs for different times (0, 24, 48, and 72h) were analysed by using 10% SDS–PAGE and immunoblotted with antibodies. Quantitative analysis of immunoblots were estimated by chemiluminescent detection. Data are presented as means ± SD (n= 3). The asterisk indicates a significant difference between control and drug-treated cells (*P < 0.05)

As illustrated in [Figure 7], compared with the control group, Bel-7402 cells treated with n-butyl-β-D-fructofuranoside resulted in an evident decline of Bcl-2 and significant increase of Bax and p53 (P < 0.05). The results revealed that n-butyl-β-D-fructofuranoside induced the upregulation of p53 expression in a time-dependent manner, which was coincidentally correlated with the upregulation of downstream effectors, such as Bax [Figures 6 and 7].

Figure 7

Real-time quantitative PCR for gene expression analysis. cDNA from hepatocellular carcinoma Bel-7402 cells samples were PCR amplified in the presence of TaqMan probes. The expressions of Bcl-2, Bax and p53 gene were compared with β-actin expression of each sample (*P < 0.05)

Discussion

The n-butyl-β-D-fructofuranoside is generally available from plants, such as: Smilax glabra,[8] Brassica rapa,[9] songaricum,[10] and easily synthesized from Bacillus subtilis,[11] Lactozyme.[12] The bioactivity and pharmacological research of the n-butyl-β-D-fructofuranoside mainly focus on transfructosylation activity,[13] and little is known about its antitumor effects. Our study on its antiproliferation activity indicated that the n-butyl-β-D-fructofuranoside significantly inhibited the proliferation of Bel-7402 cells in both time- and dose-dependent manner. Furthermore, it was assigned for further study to investigate the possible mechanism of cytotoxic effect.

Regulation and disturbance of the cancer cell cycle is a therapeutic target for development of new anticancer drugs.[14] Apoptosis has been defined as counterbalance for cell proliferation in maintaining normal tissue homeostasis.[15] In this study, it could be found that n-butyl-β-D-fructofuranoside was likely to suppress the Bel-7402 cells not only by interfering the cell cycle but also by inducing apoptosis. It is characterized by distinct morphologic changes, including cell shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies.[16] The most distinctly biochemical hallmark of apoptosis is the activation of the endogenous Ca2+/Mg2+-dependent endonuclease and endonuclease-mediated cracking nucleosomes to generate oligonucleotide fragments with about 180~200 bp length.[17] In this study, it could be found that the apoptosis of Bel-7402 cell could be induced by n-butyl-β-D-fructofuranoside associated with oligonucleosomal DNA fragmentation. A series of genes regulate the process of apoptosis. The Bcl-2 protein family is an important regulator of apoptosis, which consists of antiapoptotic (such as Bcl-2) and proapoptotic members (such as Bax),[18] when Bcl-2 heterodimerizes with Bax, it abrogates the ability of Bax and blocks apoptosis in cells.[19] In addition, p53 is the most commonly mutated gene and it is critical in the maintenance of genome integrity in that it regulates the cell cycle, DNA damage, and apoptosis, restoring apoptosis via regulation of p53.[20] The study revealed that the downregulation of Bcl-2 and the upregulation of Bax and p53 occurred at an early stage of the antiproliferative process.

The present study demonstrated that EtOH extract from Kangaisan had obvious antitumor effect on HGC, A549 and HeLa cell lines, and n-butyl-β-D-fructofuranoside was a potential antitumor compound against hepatocellular carcinoma cells by arresting the G0/G1 phase. In addition, several apoptosis signs including the appearance of sub-G1 peak, DNA fragmentation and the upregulation of Bax/Bcl-2 and p53 were discovered after the treatment of n-butyl-β-D-fructofuranoside, which suggested the possible antitumor mechanism in terms of inducing apoptosis. However, the further studies about inducing apoptosis should be carried out, while n-butyl-β-D-fructofuranoside and other potent extracts should be tested on several other cancer cell lines, the mechanism of purified compound on HGC and HeLa cells is worthy of further investigation, the biochemical as well as molecular studies are supposed to be done in both cell and animal models to establish their therapeutic efficacy and toxicity; thereby providing the scientific and credible basis in terms of the clinical use of Kangaisan.