IP6-induced growth inhibition and differentiation of HT-29 human colon cancer cells: involvement of intracellular inositol phosphates
Yang, G.Y.; Shamsuddin, A.M.
Anticancer Research 15(6b): 2479-2487
1995
ISSN/ISBN: 0250-7005 PMID: 8669811 Document Number: 454252
Inositol hexaphosphate (InsP-6 or IP-6) 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 InsP-6 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 InsP-6 and 1-6 days treatment) growth inhibition was observed as tested by MTT-incorporation assay. The inhibition was statistically significant (p lt 0.05) at 1 mM concentration as early as first day after treatment and continued lip to 6 days. DNA-synthesis was also suppressed by InsP-6 and significantly inhibited as early as 6 h after treatment at 1 mM concentration (p lt 0.05) and continued to 48 h (p lt 0.01). The expression of proliferation marker PCNA was down-regulated (p lt 0.05) by InsP-6 (1 and 5 mM) after 48 h of treatment. To investigate the mechanism of action of InsP-6, the intracellular phosphatases (including phytase) were inhibited by F- to slow down the dephosphorylation of InsP-6. Ion-exchange chromatographic separation of intracellular inositol phosphates demonstrated a 84-98% decrease of Ins, InsP-1 and InsP-2; InsP-3 was reduced by 39% and InsP-4 and InsP-5 by 21% and 13% respectively, whereas intracellular InsP-6 was increased by 24.6% at 5 min following 3H-InsP-6. Since neither the rate of uptake of 3H-InsP-6 was unaffected, not- 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 InsP-6. Alkaline phosphatase activity (brush border enzyme, associated with absorptive cell differentiation), increased following 1 and 5 mM InsP-6 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 lt 0.0001) by InsP-6. 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 InsP-6; 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 InsP-6 treatment, it can be used to monitor the efficacy of chemoprevention by InsP-6 or other such agents. Since InsP-6, 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.