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 expression 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
mucin 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 natúral 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.