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 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.
Yang
G Y
GY
Department of Pathology, University of Maryland School of Medicine, Baltimore 21201, USA.
Shamsuddin
A M
AM
D010832:6-Phytase / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000032:analysis; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology*; D014408:Biomarkers, Tumor; D002240:Carbohydrate Sequence; D002277:Carcinoma / Q000473:pathology*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003110:Colonic Neoplasms / Q000473:pathology*; D004261:DNA Replication / Q000187:drug effects; D004273:DNA, Neoplasm / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D006131:Growth Inhibitors / Q000494:pharmacology*; D006801:Humans; D007295:Inositol Phosphates / Q000502:physiology*; D007424:Intracellular Fluid / Q000378:metabolism; D008969:Molecular Sequence Data; D009077:Mucins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D010766:Phosphorylation; D010833:Phytic Acid / Q000494:pharmacology*; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D014407:Tumor Cells, Cultured / Q000187:drug effects
D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D000975:Antioxidants; D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D006131:Growth Inhibitors; D007295:Inositol Phosphates; D009077:Mucins; D009363:Neoplasm Proteins; D018809:Proliferating Cell Nuclear Antigen; D010833:Phytic Acid; D000469:Alkaline Phosphatase; D010832:6-Phytase