Effects of inositol hexaphosphate on proliferation of HT-29 human colon carcinoma cell line

Abstract

AIM: To investigate the effects of inositol hexaphosphate (IP₆) on proliferation of HT-29 human colon carcinoma cell line.

METHODS: Cells were exposed to various concen-trations (0, 1.8, 3.3, 5.0, 8.0, 13.0 mmol/L) of IP₆ for a certain period of time. Its effect on growth of HT-29 cells was measured by MTT assay. The expressions of cell cycle regulators treated with IP₆ for 2 d were detected by immunocytochemistry.

RESULTS: IP₆ inhibited the HT-29 cell growth in a dose- and time-dependent manner. Analysis of cell cycle regulator expression revealed that IP₆ reduced the abnormal expression of P53 and PCNA and induced the expression of P21.

CONCLUSION: IP₆ has potent inhibitory effect on proliferation of HT-29 cells by modulating the expression of special cell cycle regulators.

Key Words: Phytic Acid; Colonic neoplasms; Cell proliferation

INTRODUCTION

Colorectal cancer is the second most frequent cancer in Western countries[1], and the third leading cause of cancer deaths in the United States[2]. In China, the mortality rate of colorectal cancer is the fourth to sixth leading cause of cancer deaths[3]. Epidemiological studies have shown that high fiber foods, such as fruits, vegetables, whole grains and cereals, may protect against colorectal cancer[4-8]. Animal studies have shown that wheat bran has protective effect against colorectal cancer[9-14], which is attributed mostly to its high fiber content. Interestingly, many of the proposed protective mechanisms of wheat bran fiber, such as decreased transit time[15], increased bulk[16] and fermentation[17], are analogous to those of inositol hexaphosphate (IP₆ or phytic acid), which is a major fiber-associated component of wheat bran[18]. In some epidemiological studies, colorectal cancer-protective effect of fiber foods rich in IP₆, such as wheat bran has been observed[18], indicating that IP₆ may protect against colorectal cancer.

IP₆ is a naturally occurring polyphosphorylated carbohydrate, found in plants, particularly in cereals and legumes (0.4%-6.4%)[19]. It consists of a myo-inositol ring with six dihydrogen phosphate groups, assuming a chair conformation in dilute solution[20]. This unique structure empowers IP₆ with a chelating capacity of binding to polyvalent (both mono and divalent) cations. Some of these metal ions such as magnesium and zinc play an important role in stimulation of cellular proliferation[21]. This molecule is related to human health as an anti-nutrient. However, during the last decades it has been shown that IP₆ is also widely distributed in animal cells and tissues at substantial levels[22,23]. Especially, a strong anti-cancer activity of IP₆ has been demonstrated both in vivo and in vitro[24]. IP₆ exerts its anti-cancer activity by entering into cellular inositol hexaphosphate pool and affecting common cellular signal transduction pathways[24,25], but its mechanisms of action are still not completely understood.

This study was to examine the effect of IP₆ on growth of HT-29 human colon carcinoma cell line. The expressions of cell cycle regulators were assessed after IP₆ treatment.

MATERIALS AND METHODS

Chemicals

IP₆ (a dodecasodium salt from rice) and 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) were purchased from Sigma (St Louis, MO, USA). DMEM/Ham F12 culture medium, fetal bovine serum and trypsin were from Gibco BRL (Grand Island, NY, USA). Rabbit polyclonal antibody to human P53, mouse monoclonal antibodies to human P21, PCNA and SP histostain-plus kits were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell lines, culture conditions and IP₆ treatment

The HT-29 human colon carcinoma cell line was obtained from Xiehe Medical University (Beijing, China). Cells were grown in DMEM/Ham F12 medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 g/L) under standard culture conditions (37°C, 950 mL/L humidified air and 50 mL/L CO₂). A stock solution of 100 mmol/L IP₆ in distilled water was freshly prepared each time before use, the pH was neutralized with NaOH and sterilized by passing through a 0.22 μmol/L membrane filter. Dilutions of 1.8, 3.3, 5.0, 8.0, 13.0 mmol/L in DMEM/Ham F12 medium were prepared from stock solution immediately before use. The DMEM/Ham F12 medium with equal volume of distilled water served as negative control.

MTT assay

Cell number was determined by colorimetric MTT assay[24]. MTT was dissolved in PBS at 5 g/L, filter-sterilized, diluted in the DMEM/Ham F12 medium, giving a final concentration of 1.0 g/L. For growth assay, cells were plated in 96-well microtiter plates (Costar, Cambridge, MA, USA) at a density of 2 × 10³ cells/well of 100 μL media. One of the IP₆ treatments was that cells were exposed to 1.8-13.0 mmol/L IP₆ for 6 h, 12 h, or 24 h, after which IP₆ was removed and cells were grown in media without IP₆. This treatment was given every other day and continued for 6 d. The other IP₆ treatment was that cells were treated with various concentrations of IP₆ continuously for 6 d, during which culture media were changed with fresh media every other day. At the indicated time the media were removed, 50 μL of MTT was added, and the incubation was continued for 4 h at 37°C. Individual cell viability was assessed by visualization of intracellular blue crystal formation by light microscopy. The precipitated formazan was dissolved with 150 μL of DMSO, and the absorbance was determined at 490 nm with a microplate autoreader (EL311sx, Bio-Tek Instruments, Inc., Winooski, VT). Cell growth assay was repeated three times.

Immunocytochemistry

Immunocytochemical staining for P53, P21 and PCNA was carried out by the standard streptavidin-peroxidase-biotin technique (SP technique) using SP kit. Cells were treated with 1.8-13.0 mmol/L IP₆ for 2 d and collected by a brief trypsinization and plated on slides. The cells were fixed in acetone at -20°C for 5 min. The endogenous peroxidase activity was quenched in a 3% solution of hydrogen peroxide for 15 min and blocked for 10 min. Cells were immunostained with monoclonal antibody (dilutions: P21 1:50, PCNA 1:80) and P53 polyclonal antibody (dilutions: 1:50) for 1 h at 37°C. After three further washes with PBS, a second biotinylated goat anti-rabbit or rabbit anti-mouse antibodiy was applied for 1 h at room temperature and then streptavidin conjugated to peroxidase was added. Following extensive washes with PBS, 3, 3-diaminobenzidine was used for color development, and hematoxylin was used for counterstaining. The negative controls were performed by substituting the primary antibody with PBS. Hematoxylin-stained cells were examined under light microscope and photographed. Cells not counterstained were measured by VIDAS2.1 image analysis system for absorbance because hematoxylin staining could affect the image-analysis results. Three highly magnified visual fields which were not overlapped were randomly selected to measure the absorbance of each field. The mean absorbance was calculated.

Statistical analysis

The experimental results were repeated three times and expressed as mean ± SD. Statistical analysis was carried out using one-way ANOVA. P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 11.5 (SPSS Inc, Chicago, IL, USA).

RESULTS

Effect of IP₆ on the growth of HT-29 cells

Continuous treatment with IP₆ inhibited the proliferation of HT-29 cells (Figure 1A). The absorbance value for each IP₆ group was lower than that of control group. At the same time point, the absorbance value decreased with increasing IP₆ concentration. The absorbance values for the 8.0 mmol/L and 13.0 mmol/L IP₆ groups decreased on d 6 (P < 0.05). The effects of discontinuous treatment with IP₆ on the growth of HT-29 cells are shown in Figures 1B-1D. The treatment with IP₆ for 24 h inhibited the cell growth. But neither 6 h nor 12 h treatment showed dose- or time-dependent inhibition effects though the absorbance value for each IP₆ group was lower than that for control group.

Figure 1 MTT assay showing the effect of IP₆ on the growth of HT-29 cells after treated for 0 (A), 6 (B), 12 (C), 24 h (D).

Effects of IP₆ on the expression of P53, P21 and PCNA

Compared to the control, the expression of P53 protein in HT-29 cells was decreased after 2 d of IP₆ treatment at different concentrations (P < 0.05) (Table 1 and Figure 2A, Figure 2B).

Table 1 P53 expression in IP₆-treated HT-29 cells (mean ± SD).

Concentration of IP6 (mmol/L)	Absorbance value
Control	0.6772 ± 0.0095
1.8	0.6161 ± 0.0203
3.3	0.5996 ± 0.0205
5.0	0.6067 ± 0.0130
8.0	0.5871 ± 0.0159
13.0	0.5817 ± 0.0158

Figure 2 Expression of p53 (A, B), p21 (C, D) and PCNA (E, F) in untreated control and IP₆-treated cells. The slides were counterstained with hematoxylin (× 400, × 100,× 400, respectively).

Compared to the control, treatment of HT-29 cells with IP₆ at various concentrations for 2 d increased the expression of P21 (P < 0.05) (Table 2 and Figure 2C, Figure 2D).

Table 2 P21 expression in IP₆-treated HT-29 cells (mean ± SD).

Concentration of IP6 (mmol/L)	Absorbance value
Control	0.4868 ± 0.0486
1.8	0.6011 ± 0.0152
3.3	0.5138 ± 0.0336
5.0	0.6032 ± 0.0105
8.0	0.6078 ± 0.0066
13.0	0.5981 ± 0.0163

Compared to the control, the expression of PCNA decreased after treated with IP₆ at different concentrations for 2 d (P < 0.05) (Table 3 and Figure 2E, Figure 2F).

Table 3 PCNA expression in IP₆-treated HT-29 cells (mean ± SD).

Concentration of IP6 (mmol/L)	Absorbance value
Control	0.6407 ± 0.0096
1.8	0.6361 ± 0.0087
3.3	0.5904 ± 0.0302
5.0	0.4520 ± 0.0495
8.0	0.4788 ± 0.0357
13.0	0.5006 ± 0.0403

The absorbance values assayed by MTT after IP₆ treatment for different periods of time are listed in Table 4, Table 5, Table 6, and Table 7.

Table 4 Absorbance values assayed by MTT after continuous treatment with IP₆ (mean ± SD).

IP6 (mmol/L)	Time (d)
	0	2	4	6
Control	0.0847 ± 0.0021	0.1487 ± 0.0031	0.3083 ± 0.0240	0.3450 ± 0.0056
1.8	0.0857 ± 0.0045	0.1293 ± 0.0049	0.2320 ± 0.0155	0.3097 ± 0.0180
3.3	0.0837 ± 0.0042	0.1203 ± 0.0075	0.1770 ± 0.0227	0.2537 ± 0.0300
5.0	0.0873 ± 0.0057	0.1233 ± 0.0029	0.1547 ± 0.0042	0.2077 ± 0.0153
8.0	0.0857 ± 0.0021	0.0940 ± 0.0053	0.0873 ± 0.0032	0.0757 ± 0.0025
13.0	0.0847 ± 0.0032	0.0660 ± 0.0010	0.0530 ± 0.0026	0.0510 ± 0.0036

Table 5 Absorbance values assayed by MTT after IP₆ treatment for 6 h (mean ± SD).

IP6 (mmol/L)	Time (d)
	0	2	4	6
Control	0.0847 ± 0.0021	0.1487 ± 0.0031	0.3083 ± 0.0240	0.3450 ± 0.0056
1.8	0.0857 ± 0.0045	0.1397 ± 0.0023	0.2823 ± 0.0344	0.3303 ± 0.0080
3.3	0.0837 ± 0.0042	0.1333 ± 0.0015	0.2317 ± 0.0192	0.3383 ± 0.0025
5.0	0.0873 ± 0.0057	0.1277 ± 0.0025	0.2593 ± 0.0131	0.3240 ± 0.0082
8.0	0.0857 ± 0.0021	0.0940 ± 0.0053	0.0873 ± 0.0032	0.0757 ± 0.0025
13.0	0.0847 ± 0.0032	0.1157 ± 0.0035	0.2237 ± 0.0215	0.3020 ± 0.0080

Table 6 Absorbance values assayed by MTT after IP₆ treatment for 12 h (mean ± SD).

IP6 (mmol/L)	Time (d)
	0	2	4	6
Control	0.0847 ± 0.0021	0.1487 ± 0.0031	0.3083 ± 0.0240	0.3450 ± 0.0056
1.8	0.0857 ± 0.0045	0.1383 ± 0.0065	0.2660 ± 0.0056	0.3267 ± 0.0050
3.3	0.0837 ± 0.0042	0.1230 ± 0.0043	0.1990 ± 0.0554	0.3220 ± 0.0105
5.0	0.0873 ± 0.0057	0.1250 ± 0.0017	0.2290 ± 0.0350	0.3217 ± 0.0076
8.0	0.0857 ± 0.0021	0.1103 ± 0.0038	0.2693 ± 0.0711	0.2723 ± 0.0025
13.0	0.0847 ± 0.0032	0.0957 ± 0.0035	0.2363 ± 0.0299	0.2107 ± 0.0135

Table 7 Absorbance values assayed by MTT after IP₆ treatment for 24 h (mean ± SD).

IP6 (mmol/L)	Time (d)
	0	2	4	6
Control	0.0847 ± 0.0021	0.1487 ± 0.0031	0.3083 ± 0.0240	0.3450 ± 0.0056
1.8	0.0857 ± 0.0045	0.1323 ± 0.0040	0.2547 ± 0.0153	0.3330 ± 0.0122
3.3	0.0837 ± 0.0042	0.1277 ± 0.0099	0.2300 ± 0.0161	0.3123 ± 0.0115
5.0	0.0873 ± 0.0057	0.1317 ± 0.0032	0.2137 ± 0.0168	0.2990 ± 0.0190
8.0	0.0857 ± 0.0021	0.1067 ± 0.0021	0.1757 ± 0.0116	0.2093 ± 0.0105
13.0	0.0847 ± 0.0032	0.0807 ± 0.0021	0.0650 ± 0.0036	0.0857 ± 0.0051

DISCUSSION

Uncontrolled proliferation is one of the most important characteristics of malignant cells due to the aberrations of cell cycle regulators such as mutation, activation or inactivation of genes. Identification of cell cycle regulator specificity of anti-tumor drugs is essential to understand the mechanisms of their action.

MTT assay in this study showed that the growth of HT-29 cells was inhibited after continuous IP₆ treatment for 2-6 d (P < 0.05). The effect enhanced with increasing IP₆ concentration and prolonged treatment time, suggesting that the inhibition effects of IP₆ are dose- and time-dependent.

To confirm our data we used another proliferating marker, proliferating cell nuclear antigen (PCNA) which is essential for both DNA replication and repair[26]. During DNA replication, PCNA forms a ring structure clamping the synthesized DNA to the DNA polymerases δ and ε to ensure continuation of the replication process[27,28]. In case of DNA damage, PCNA binds to the over-expressed P21^waf1/cip1 leading to inhibition of PCNA-dependent replication, but it does not affect the DNA repair function attained by PCNA[29]. Thus, PCNA is expressed in both cycling and non-cycling cells[30]. Immunocytochemistry in this study showed that IP₆-treated cells reduced PCNA expression compared with control cells (P < 0.05), although the dose-dependent inhibition was not obvious, which was in agreement to the low proliferation rate observed in MTT assay, indicating that IP₆ inhibits proliferation of HT-29 cells.

Since IP₆ inhibits cell growth, we studied the regulators of cell cycle. P53, a tumor suppressor protein, is a nuclear transcription factor that controls cell cycle progression[31,32], and plays a role in G1/S check point of cell cycle allowing the repair of damaged DNA[33,34]. Mutations and deletions of the tumor suppressor gene p53 have been identified in about 50% of colorectal carcinomas and are associated with poor prognosis due to its weaker ability to inhibit cell proliferation. The half-life of wild-type P53 is very short and difficult to detect, while the mutant P53 protein has a much longer half life and can be examined by conventional immunohistochemical technology[35]. Rodrigues NR et al[36] showed that over-expression of p53 is synonymous with mutation and HT-29 cells have mutations in codon 273 of the p53 gene, so HT-29 cells overproduce mutant p53 antigen. In our study, the immunocytochemical results showed that in IP₆-treated cells the abnormal expression of P53 protein decreased compared to control (P < 0.05), indicating that IP₆ reduces the expression of mutant P53 protein. It was reported that treatment of HT-29 cells with IP₆ increases the level of wild-type P53[37]. In the present study, P53 polyclonal antibody was not specified for wild P53 but responded to many antigenic determinants, including mutant P53, indicating that IP₆ up-regulates the expression of wild-type P53 and down-regulates the expression of mutant P53 to control cell cycle check-point and prevent progression of cells to the DNA synthesis phase (S phase) of the cell cycle. But the exact mechanism by which IP₆ affects p53 is not clear and needs further study.

P21^waf1/cip1 is an inhibitor of cyclin dependent kinases (CDKS) that are required for the cells to enter the S-phase of the cell cycle[38]. The gene encoding P21^waf1/cip1 is transcriptionally regulated by the protein product of the gene p53. Over-expression of P21^waf1/cip1 is growth inhibitory, possibly by inhibiting the activity of cyclin/CDK complex[39] which binds to the C-terminal domain of PCNA. The resulting P21-PCNA complex blocks the ability of PCNA to process DNA polymerase in DNA replication. Thus P21^waf1/cip1 may act as a tumor suppressor because of its role in growth control[39,40]. In the present study, the expression of P21 was increased after IP₆ treatment for 2 d (P < 0.05). After counterstaining with hematoxylin, untreated cells were stained purple while IP_6-treated cells were stained yellow, indicating that expression of P21 is higher in IP₆-treated cells. High-expression of P21^waf1/cip1 leads to decreased nuclear expression of PCNA, which is in agreement with our results.

In summary, IP₆ remarkably inhibits proliferation of HT-29 human colon carcinoma cell line. IP₆ exerts its inhibitory effect in part by affecting special cell cycle regulators and reduces over-expression of mutant P53 and stimulates expression of wild-type P53 and P21^waf1/cip1. P21^waf1/cip1 binds to PCNA, thus preventing PCNA-dependent cellular proliferation. In our immunocytochemical experiments, cells grew very slowly and were not adhered in media with high IP₆ dose, fell off and died very soon. The effect of 13.0 mmol/L IP₆ on expression of genes was less than that of 8.0 mmol/L IP₆, partly due to the rapid death of cells in 13.0 mmol/L IP₆, indicating that that IP₆ has no significant effect on the expression of genes. Furthermore, neither significant dose-dependent effect of IP₆ was observed on the expressions of cell cycle regulators nor obvious correlation among these indexes was found, possibly owing to the short period of IP₆ treatment (only 2 d), suggesting that the effects of IP₆ on gene expressions are relatively weak.

The present study is merely a preliminary investigation of IP₆ on colon cancer. The results are also limited although the effects of IP₆ can be seen. Additional research is needed to explore the mechanisms of IP₆ in cell proliferation and differentiation, apoptosis, and potential therapeutic value of IP₆.