Apoptosis is induced by the active metabolite of vitamin D-3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: Possible implicationsfor prevention and therapy

Vitamin D-3 is believed to reduce the risk of colon cancer, and serum level s inversely correlate with colorectal cancer incidence. The active metaboli te, 1 alpha,25-dihydroxyvitamin D-3, has previously been shown to inhibit g rowth and promote differentiation of colon cancer cells. The vitamin D anal ogue, EB1089, is currently under clinical trial in a variety of cancers bec ause of its growth-inhibitory effects in vitro and reduced hypercalcemic ef fects in vivo. The mechanism of growth inhibition by EB1089, however, remai ned to be determined. In this study we examined the effects of la,25-dihydr oxyvitamin D-3 and EB1089 on five colorectal tumor cell lines (two adenoma and three carcinoma) to determine the mechanism of growth inhibition and to ascertain whether premalignant adenoma cells were responsive to these agen ts. 1 alpha,25-Dihydroxyvitamin D, and EB1089 induced p53-independent apopt osis in adenoma and carcinoma cell lines in a dose-dependent manner between 10(-10) and 10(-6) M. EB1089, as web as inducing apoptosis, increased the proportion of cells in the G(1) phase, particularly in the adenoma cell lin es. In two of the three carcinoma cell lines (SW620 and PC/JW), levels of a poptosis induced by EB1089 were similar or greater than those induced by 1 alpha,25-dihydroxyvitamin D-3. Although the carcinoma cell line HT29 was re latively resistant to apoptosis induced by EB1089 compared with 1 alpha,25- dihydroxyvitamin D-3. EB1089 markedly inhibited cell yields. These observat ions offer promise fur the clinical use of EB1089. To determine whether the induction of apoptosis by 1 alpha,25-dihydroxyvitamin D-3 and EB1089 was p otentially via a differentiation pathway, alkaline phosphatase activity was measured as a marker of differentiation. Induction of alkaline phosphatase was observed in the floating apoptotic cells as well as in the adherent po pulation. A link between the induction of differentiation and apoptosis by 1 alpha,25-dihydroxyvitamin D-3 and EB1089 is suggested by the occurrence o f apoptosis subsequent to the induction of differentiation. To investigate the molecular pathway to apoptosis induction, members of the Bcl-2 family o f proteins were examined (Bcl-2, Bcl-x, Bax, and Bak). Decreased Bcl-2 was observed in some cell lines, particularly in response to EB1089, but was no t essential for apoptosis. Levels of the proapoptotic protein bah, however, were consistently increased in all of the five cell Lines in association w ith apoptosis induced by either agent. The results implicate Bak protein in the induction of apoptosis by 1 alpha,25-dihydroxyvitamin D, or its analog ue EB1089. The ability of EB1089 to induce apoptosis in colorectal carcinom a cells suggests that this or other vitamin D analogues may prove clinicall y effective for the treatment of colorectal cancer. Furthermore, the fact t hat it induces cell cycle arrest and apoptosis in the premalignant adenoma cells may suggest an application in colorectal cancer chemoprevention.