IP6-INDUCED GROWTH-INHIBITION AND DIFFERENTIATION OF HT-29 HUMAN COLON-CANCER CELLS - INVOLVEMENT OF INTRACELLULAR INOSITOL PHOSPHATES

Inositol hexaphosphate (InsP(6) or IP6) ubiquitous in plants and anima ls is not only a natural antioxidant, but may also be the precursor/st orage of intracellular inositol phosphates, important for various cell ular 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 col on carcinoma cells in vitro. A dose- and time-dependent (0.33-20 mM In sP(6) and 1-6 days treatment) growth inhibition was observed as tested bit MTT- incorporation assay. The inhibition was statistically signif icant (p < 0.05) at 1 mM concentration as early as first day after tre atment 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 < 0.05) and continued to 48 h (p < 0.01). Th e expression of proliferation marker PCNA was down-regulated (p < 0.05 ) by InsP(6) (1 and 5 mM after 48 h of treatment. To investigate the m echanism of action of InsP(6), the intracellular phosphatases (includi ng phytase) were inhibited by F- to slow down the dephosphorylation of InsP(6). Ion-exchange chromatographic separation of intracellular ino sitol phosphates demonstrated a 84-98% decrease of Ins, InsP(1) and In sP(2); InsP(3) was reduced by 39% and InsP(4) and InsP(5) by 21% and 1 3% respectively, whereas intracellular InsP(6) was increased by 24.6% at 5 min following H-3-InsP(6). Since neither the late of uptake of H- 3-InsP(6) was unaffected nor was the efficacy of growth inhibition alt ered by F- inhibition of phytase, data suggest that contrary to the po pular misconception, phytase plays no role in influencing the anti-neo plastic action of InsP(6). Alkaline phosphatase activity (brush border enzyme, associated with absorptive cell differentiation), increased f ollowing 1 and 5 mM InsP(6) treatment for 1-6 days. The expression of a mucin antigen associated with goblet cell differentiation and define d by the monoclonal antibody CMU10 was augmented (p < 0.0001) by InsP( 6). The tumor mucin marker Gal-GalNAc, expressed by precancer and canc er of colon, but not by the normal cells showed a time-dependent bipha sic change by InsP(6); an increased expression after 1 day of treatmen t followed by suppression after 2 days suggest progression of mucin sy nthesis and differentiation of cancer cells with reversion to normal p henotype. Because the tumor marker Gal-GalNAc is a) easily detected in rectal mucin of patient with colonic cancer and precancer with high s ensitivity 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 cere als and legumes, inhibits growth and induces terminal differentiation of HT-29 cancer cells, it is an excellent candidate for adjuvant chemo therapy and prevention of cancer.