Introduction

Phytic acid (InsP6, myo-inositol hexaphosphoric acid, see Fig. 1) is present in substantial amounts in plant seeds, comprising 1–5% by weight in edible legumes, cereals, oil seeds, pollens, and nuts (Reddy and Sathe, 2002). The six phosphate groups of InsP6 have strong chelating capacity to essential divalent cations, such as calcium, magnesium, iron, zinc, and manganese, forming largely insoluble complex, and thereby decreasing their nutritional bioavailability, so InsP6 has long been considered to be a kind of anti-nutrient (Harland and Morris, 1995; Yoon et al., 1983). However, the value of InsP6 in foods to prevent and possibly reverse carcinogenesis is now recognized. Rodent models showed that InsP6 was effective in preventing cancer of the colon (Barrett et al., 1998), prostate (Singh and Agarwal, 2005), lung (Sun et al., 2001), skin (Gupta et al., 2003), and liver (Vucenik et al., 1998). The anticancer action of InsP6 was reviewed by Shamsuddin (2002).

Fig. 1
figure 1

The structure of inositol (a), phytic acid (b), and Ins(1,2,6)P3 (c), Pi means –PO(OH)2

Full size image

The partial hydrolysis of InsP6 produces different inositol phosphates by phytases or thermal process. The second messenger role of inositol 1,4,5-triphosphate (Ins(1,4,5)P3) has well established. A lot of other lower inositol phosphates (InsP1–4) are present in most mammalian cells regulating vital cellular functions. InsP6 undergoes dephosphorylation to InsP1–5, and among which InsP3 is central in cellular signal transduction and intracellular function. Whether Ins(1,2,6)P3, the hydrolysis product of phytic acid, can inhibit cancer cell proliferation is unclear. Therefore, Ins(1,2,6)P3 was prepared and applied on the human liver cancer cell SMMC-7721 for the first time.

Results and discussion

Inhibitory effects of Ins(1,2,6)P3 on SMMC-7721 cells proliferation

The inhibitory effects of Ins(1,2,6)P3 (50, 100, 300, 500, and 700 μg/ml) on SMMC-7721 cells proliferation is presented in Table 1 (data for 24 and 48 h treatment are not shown). It showed that Ins(1,2,6)P3 had significant inhibitory effect on SMMC-7721 cells proliferation and was in a dose and time-dependent manner. For example, after treatment with 700 μg/ml Ins(1,2,6)P3 for 72 h, the inhibition ratio reached 79.08% (P < 0.01).

Table 1 Effects of Ins(1,2,6)P3 on proliferation of SMMC-7221 after 72-h treatment
Full size table

Effects of Ins(1,2,6)P3 on morphology of SMMC-7721 cells

SMMC-7721 cells in the control group were epithelioid, most of which were spindle or kidney-shaped (Fig. 2a), while among Ins(1,2,6)P3-treated cells (Fig. 2b), the killing effect is obviously occurred, in which many cancer cells were detached from matrix, and cell structure were in disruption.

Fig. 2
figure 2

Morphological observation of SMMC-7721 cells treated for 48 h with Ins (1,2,6)P3. The cultured SMMC-7721 cells were stained with HE. a Control and b after treatment with 300 μg/ml Ins(1,2,6)P3. The cells were observed in Olympus BH-2 optical microscope with ×200 magnification

Full size image

Effects of Ins(1,2,6)P3 on SMMC-7721 cells cycle

The cell cycle phase distribution of SMMC-7721 cells after treatment with Ins(1,2,6)P3 is summarized in Table 2 (the original information see supporting information). Ins(1,2,6)P3 treatment induced an accumulation of cells in the G0/G1 phase of the cell cycle. For example, 500 μg/ml treatment for 3 days resulted in an increase in the percentage of cells in the G0/G1 phase from 39.0 to 68.1%. Concomitant with this increase, the percentage of cells in the S phase was significant decreased from 49.5 to 12.3%. So Ins(1,2,6)P3 induced a block at the G0/G1 phase. Therefore, our data indicated that Ins(1,2,6)P3 could change cell cycle of SMMC-7721 cells to take anticancer action.

Table 2 Effects of Ins(1,2,6)P3 on cell cycle of SMMC-7721 cells
Full size table

Effects of AFP and PCNA of SMMC-7721 cells

AFP expression of SMMC-7721 cells

AFP expression serves as an important sign of the malignant degree of tumor cells and has close correlation with the differentiation ability of cells. As shown in Fig. 3, SMMC-7721 cells in control group demonstrated a stronger AFP expression, with yellowish-brown color mainly in the area of cytoplasm as well as nucleolus. After treatment with 300 and 500 μg/ml Ins(1,2,6)P3 for 48 h, the protein expression in nucleolus and cytoplasm reduced distinctly, which the color changed into buff. A dose–effect relationship was also observed. In other words, α-fetal protein (AFT) expression decreased with the increase of Ins(1,2,6)P3 concentration, which shows that Ins(1,2,6)P3 had inhibitory effects on the AFP expression of SMMC-7721 cells and facilitated the SMMC-7721 cell differentiation. The malignant transformation of cells means the unbalance between cell proliferation and cell differentiation. Cancer cell proliferation is uncontrolled in the aspect of cell proliferation, or cancer cells lose differentiation or with abnormal differentiation in the aspect of differentiation. In this respect, Ins(1,2,6)P3 facilitated the SMMC-7721 cell differentiation to exert anticancer activities.

Fig. 3
figure 3

Effects of Ins(1,2,6)P3 on the alpha-fetoprotein (AFP) expression in SMMC-7721 cells. The SMMC-7721 cells were treated with Ins(1,2,6)P3 for 48 h. The chromogenic reaction was developed with DAB. a control, b 300 μg/ml Ins(1,2,6)P3, and c 500 μg/ml Ins(1,2,6)P3. The cells were observed in Olympus BH-2 optical microscope with ×400 magnification

Full size image

PCNA expression of SMMC-7721 cells

PCNA is an important parameter of cell proliferation speed. Expression of PCNA will remarkably boost up when the tumor cell proliferation becomes active and is in the malignant direction. The test results of PCNA are shown in Fig. 4. In the control group, PCNA has a strong expression in SMMC-7721 cells, with yellowish-brown color mainly distributed in the nucleolus. After treatment with 300 and 500 μg/ml Ins(1,2,6)P3 for 48 h, the PCNA expression of nucleolus displayed an evident reduction which the color changed into buff. Once again, the dose–effect relationship was also observed. Ins(1,2,6)P3 can reduce the expression of PCNA of SMMC-7721 cells, which was in line with the MTT results.

Fig. 4
figure 4

Effects of Ins(1,2,6)P3 on the PCNA expression in SMMC-7721 cells. The SMMC-7721 cells were treated with Ins(1,2,6)P3 for 48 h. The chromogenic reaction was developed with DAB. a Control, b 300 μg/ml Ins(1,2,6)P3, and c 500 μg/ml Ins(1,2,6)P3. The cells were observed in Olympus BH-2 optical microscope with ×200 magnification

Full size image

In conclusion, the proliferation of SMMC-7721 cancer cells can be inhibited by Ins(1,2,6)P3 and change the cell cycle by blocking the cells at G0/G1 phase. AFP test indicated that Ins(1,2,6)P3 can inhibit the malignant expression and proliferation of SMMC-7721 cells by inducing the cells to differentiate toward normal cells. From this point, the partial hydrolysate product of phytic acid may provide beneficial to health while its strong anti-nutrient effect is prevented because its metal ion binding ability is decreased for its less phosphate groups.

Experiment

Materials and methods

Trypsinase and RPMI-1640 were supplied by Gibco BRL. Dimethyl sulphoxide (DMSO), [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) were purchased from Amresco Co. Ribonuclease A (RNase A), propidium iodide (PI) and phytic acid were obtained from Sigma-Aldrich Chemical Co. Goat serum, biotinylated anti-mouse IgG, proliferating cell nuclear antigen (PCNA), and mouse-anti-human AFT were obtained from Wuhan Sanli Biotechnology Co. Other products were purchased from Chinese Reagent Co.

Preparation of Ins(1,2,6)P3

Ins(1,2,6)P3 was prepared as described by Phillippy with some modifications (Phillippy et al., 1987). Sodium phytate (25.0 g) was dissolved in 200 ml 0.1 mol/l HAc/NaAc (pH 5.1) solution. Aspergillus terreus CCTCCAF93044 phytase (700 U) was added and the solution was diluted to 500 ml with 0.1 mol/l HAc/NaAc (pH 5.1). The solution was then incubated at 55°C. When the inorganic phosphorus liberated from phytate was about 50% of the total phosphorus contained in InsP6 after about 2 h incubation, the reaction was stopped by adding 1 mol/l ammonium water to pH > 7.0. Inorganic phosphate was assayed by molybdate blue method. The hydrolysate was filtrated. The solution was pumped into a 2- * 35-cm glass column containing 717 type strong anion resins. A linear gradient of 0.05–0.7 mol/l HCl 2,000 ml was applied to elute the inositol phosphates at a flow rate of 100 ml/h. Tubes of 10 ml were collected at 6-min interval. The pure fractions of InsP3 were collected together and the solvent was removed by rotary evaporation under vacuum at 40°C until only a dry residue remained; 20 ml of water was used to redissolve the residue and the pH was adjusted to 7.0 by slowly adding 1 mol/l NaOH. By this method, 8.2 g sodium of InsP3 was produced. The structure of the product was determined by IR (AVATAR 330 Infrared Spectrometer), 1H NMR (Bruker AV 300-MHz H Nuclear Magnetic Resonance Spectrometer), MS (Saturn 2200 Mass Spectrograph). The results were as follows: 1H NMR (δ, D2O): 4.72–4.70 (1H, d, J = 6.0 Hz), 4.32–4.22 (1H, m), 3.99–3.92 (1H, t, J = 7.0), 3.87–3.80 (1H, t, J = 7.0), 3.54–3.44 (2H, m). MS(m/z, %): 552.7 ((InsP3Na6 + 1)+,48). IR (KBr) υ, cm−1: 3453.53 (–OH), 1643.11 (O–H), 1116.12 (C–O), 975.67 (P–O).

Cell cultivation

Human liver cancer SMMC-7721 cell line was purchased from Wuhan University. Cells were grown in 75-cm2 flask with RPMI-1640 medium (containing 20% calf serum, 100 μ/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml kanamycin) at 37°C, 5% CO2, and saturated humidity. Cells grew adhering to the inner wall of flask by single layer. For passage, cells were treated with 0.25% trypsin and made into suspension with RPMI-1640 medium in advance. The cells were cultured for 24 h before the treatments. In MTT assay, SMMC-7721 cells were plated in 96-well culture plates (3 × 104 cells per well). Then cells were treated with Ins(1,2,6)P3 (0, 50, 100, 300, 500, and 700 μg/ml) for 1, 2, and 3 days. The medium was replaced every other day. The solvent control was PBS in culture medium. While in other experiments, SMMC-7721 cells were cultured with basal medium and medium containing specific concentration of Ins(1,2,6)P3.

Cell proliferation assay

Inhibition of cell proliferation by Ins(1,2,6)P3 was measured by MTT assay. After treatment of cells, 5 mg/ml MTT solution (10 μl) was added to each well for additional 4 h incubation at 37°C. After adding stop solution of DMSO (100 μl/well), the absorbance was read with a microplate reader (Multiskan MK3, USA) at 570 nm. The background control was performed in parallel. The results were expressed as the inhibition of proliferation. The inhibition ratio was calculated as follows: (1 − A sample/A control) × 100%. The data represented the change in absorbance values at 570 nm relative to that obtained from the control group. Assays were repeated at least three times.

Morphological observation

Each well of a 6-well plate was loaded with a piece of sterile coverslip and SMMC-7721 cells were seeded at a density of 1 × 105 cells/well into 6-well plates. After 24 h adherence, the cells were treated with 300 μg/ml Ins(1,2,6)P3 for 48 h. Then the coverslips were take out and washed with D-Hank’s solution, followed by fixation with Bouin-Hollande overnight. The cells were examined with Olympus BH-2 optical microscope after HE staining.

Cell cycle analysis by flow cytometry

The perturbations in the distribution of cells in the different phases of the cell cycle were determined by flow cytometry in SMMC-7721 cells after 3 days exposure to 100, 300, and 500 μg/ml Ins(1,2,6)P3, respectively. Cells were collected by trypsinization, washed by PBS, and were fixed with ice-cold 70% ethanol for at least 24 h. The cells were centrifuged at 1,000 rpm for 10 min twice, and washed by PBS. The cells were resuspended in 500 μl RNase A (1 g/l) and incubated at 37°C for 30 min. The reaction was stopped by putting them in ice for 2 min. Then cells were stained with 500 μl PI (100 mg/l). The samples were analyzed by flow cytometry (Beckman EPICS ALTRA II, USA) after putting them in the dark at 4°C for 2 h. The percentage of cell population at a particular phase was estimated with Multicycle for Windows Analysis software. Flow cytometry equipped with a 15 mw argon laser at 488 nm was used. Fluorescence intensity of PI was detected at emission wavelength 630 nm.

Immunohistochemistry

After culture for 24 h in 24-well plates (1 × 105 cells/well), the supernatant was replaced by 100 μl culture medium supplemented with Ins(1,2,6)P3 (300 and 500 μg/ml). The medium was discarded after 2 days. Ice-cold acetone was added for 10 min fixation at 4°C. Then fixation solution was discarded and PBS was added to wash the cells for three times. Immerse the cells in 3% hydrogen peroxide for 10 min, and then wash with water, PBS. The cells were incubated for 10 min with normal goat serum in PBS to block nonspecific binding. The cells were subsequently incubated overnight at 4°C with the relevant antibodies. The following day, the cells were incubated with biotinylated anti-mouse IgG for 15 min at 37°C, and then incubated with peroxidase-conjugated streptavidin. The chromogenic reaction was developed with DAB. The monoclonal antibodies used were mouse-anti-human PCNA, mouse-anti-human AFT. The cells were examined with Olympus BH-2 optical microscope.

Statistical analysis

The results were expressed as mean ± standard deviation. The difference between control and Ins(1,2,6)P3-treated cells was evaluated using Duncan test. P value <0.01 was considered statistically significant.