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The effect of inositol hexaphosphate on NFkB expression in human colon cancer cells

Małgorzata Kapral ,  Beata Parfiniewicz ,  Agnieszka Zachacz ,  Krzysztof Cholewa ,  Ludmiła Węglarz 

Departament of Biochemistry, Medical University of Silesia, Narcyzow 1, Sosnowiec 41-200, Poland


Phytic acid, a hexaphosphorylated inositol (IP6) is a major fiber-associated component of wheat bran and legumes. A number of recent studies revealed strong anti-cancer activity of IP6, and hence, the molecular mechanisms of its action are still under investigation. IP6 is hypothesized to target cancer through multiple pathways, i.e., modulation of cell signal transduction, inhibition of cell proliferation, and cell cycle progression, and activation of apoptosis, and induction of cell differentiation. IP6 may also be involved in many nuclear processes, including DNA repair, transcriptional regulation and mRNA transport.

Nuclear factor kB (NF-kB), a member of transcriptional factors, plays an important role in regulation of the expression of genes employed in these processes. It has been shown that NF-kB is constitutively activated in several types of tumors including colorectal cancer.

The aim of this study was to evaluate the influence of IP6 on the expression of genes encoding p65 and p50 subunits of NF-kB and of its inhibitor IkBα in human colorectal cancer cell line Caco-2.

The cells cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicilline and 100 μg/ml streptomycin. They were grown at 37°C as monolayers in a humidified atmosphere containing 5% CO2. Cells were treated with 1, 2.5, 5 mM IP6 for 1, 6, 12 and 24 h. Total RNA was extracted from control and IP6 treated cells with the use of TRIZOL® reagent according to the producer’s protocol. Quantification of the genes expression was performed by real time QRT-PCR with a SYBR Green I chemistry (SYBR Green Quantitect RT-PCR Kit, Qiagen) using an Opticon™ DNA Engine Sequence Detector (MJ Research, USA). The results were recalculated per mg of total RNA. Statistical analysis was performed with the use of Statistica 6.0 software.

Experimental data revealed time dependent changes in transcriptional activities of IκBα and p65 occurring in both control cells and in cells incubated with IP6. The expression of IκBα inhibitor did not show any significant changes in response to 1.0, 2.5, and 5 mM IP6 at 1 h incubation, compared to the expression of this gene in the control cells (p=0,7353; ANOVA). Treatment of cells with 5 mM of IP6 resulted in strong increase in IκBa expression observed at 6h (p=0,0009; Tukey test), 12h (p=0,0002) and 24h (p=0,0011). The level of p65 transcript after 1 h was lower in the cells exposed to 1, 2.5, and 5 mM IP6 than in the control cells (p<0,05; Tukey test). There was no statistically significant difference between p65 mRNA quantities in the cells exposed to increasing concentrations of IP6 for 1 h. However, the increase in transcriptional activity of p65 gene in response to 5 mM IP6 after 6h (p=0,0009) and 12 h (p=0,048) was observed. Cells treated for 24h with 2.5 mM (p=0,0026) and 5 mM IP6 (p=0,0112) showed a significant decrease in expression of p65 gene. There were no quantitative changes in the p50 gene expression in the cells treated with IP6 compared to the control cells (p>0,05; ANOVA). High correlation between the expression of IκBα and p65 (R=0,72, p=0,000; Spearman’s rank correlation test) was observed. There were any correlations between IκBα and p50 (R=0,37, p=0,1465) as well as between p50 and p65 transcript levels (R=0,39, p=0,1220).

In summary, the findings of this study show that IP6 alters p65 and Ik genes expression in colon cancer cells. Changes in transcriptional activities of Ik and p65 depend on IP6 concentration and time of interaction. These results suggest that the ability of 5 mM phytic acid to inhibit colon cancer cells proliferation may be mediated through the stimulation of IκBα expression at the mRNA level.


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Submitted: 2008-03-11 10:26
Revised:   2009-06-07 00:48