IP₆-Induced growth inhibition and differentiation of HT-29 human colon cancer cells: Involvement of intracellular inositol phosphates

Inositol hexaphosphate (InsP₆ or IP₆ ubiquitous in plants and animals is not only a natural antioxidant, but may also be the precursor/storage of intracellular inositol phosphates, important for various cellular functions. A novel anti-tumor action of InsP₆ was demonstrated in models of experimental colon and mammary carcinogenesis in vivo. We now show its effects on growth and differentiation of HT-29 human colon carcinoma cells in vitro. A dose- and time-dependent (0.33-20 mM InsP₆ and 1-6 days treatment) growth inhibition was observed as tested by MTT- incorporation assay. The inhibition was statistically significant (p < 0.05) at 1 mM concentration as early as first day after treatment and continued up to 6 days. DNA-synthesis was also suppressed by InsP₆ and significantly inhibited as early as 6 h after treatment at 1 mM concentration (p < 0.05) and continued to 48 h (p < 0.01). The expression of proliferation marker PCNA was down-regulated (p < 0.05) by InsP₆ (1 and 5 mM) after 48 h of treatment. To investigate the mechanism of action of InsP₆, the intracellular phosphatases (including phytase) were inhibited by F^- to slow down the dephosphorylation of InsP₆. Ion-exchange chromatographic separation of intracellular inositol phosphates demonstrated a 84-98% decrease of Ins, InsP₁ and InsP₂; InsP₃ was reduced by 39% and InsP₄ and InsP5 by 21% and 13% respectively, whereas intracellular InsP₆ was increased by 24.6% at 5 min following ³H-InsP₆. Since neither the rate of uptake of ³H-InsP₆ was unaffected, nor was the efficacy of growth inhibition altered by F^- inhibition of phytase, data suggest that contrary to the popular misconception, phytase plays no role in influencing the anti-neoplastic action of InsP₆. Alkaline phosphatase activity (brush border enzyme, associated with absorptive cell differentiation), increased following 1 and 5 mM InsP₆ treatment for 1-6 days. The expression of a mucin antigen associated with goblet cell differentiation and defined by the monoclonal antibody CMU10 was augmented (p < 0.0001) by InsP₆. The tumor mucin marker Gal-GalNAc, expressed by precancer and cancer of colon, but not by the normal cells showed a time-dependent biphasic change by InsP₆; an increased expresion after 1 day of treatment followed by suppression after 2 days suggest progression of mucin synthesis and differentiation of cancer cells with reversion to normal phenotype. Because the tumor marker Gal-GalNAc is a) easily detected in rectal of patients with colonic cancer and precancer with high sensitivity and specificity, and b) suppressed by InsP₆ treatment, it can be used to monitor the efficacy of chemoprevention by InsP₆ or other such agents. Since InsP₆, a natural dietary ingredient of cereals and legumes, inhibits growth and induces terminal differentiation of HT-29 cancer cells, it is an excellent candidate for adjuvant chemotherapy and prevention of cancer.