Vitamin D3 is believed to reduce the risk of
colon cancer, and serum levels inversely correlate with
colorectal cancer incidence. The active metabolite, 1alpha,25-dihydroxyvitamin D3, has previously been shown to inhibit growth and promote differentiation of
colon cancer cells. The
vitamin D analogue,
EB1089, is currently under clinical trial in a variety of
cancers because of its growth-inhibitory effects in vitro and reduced hypercalcemic effects in vivo. The mechanism of growth inhibition by
EB1089, however, remained to be determined. In this study we examined the effects of alpha,25-dihydroxyvitamin D3 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 agents. 1alpha,25-Dihydroxyvitamin D3 and
EB1089 induced p53-independent apoptosis in
adenoma and
carcinoma cell lines in a dose-dependent manner between 10(-10) and 10(-6) M.
EB1089, as well as inducing apoptosis, increased the proportion of cells in the G1 phase, particularly in the
adenoma cell lines. In two of the three
carcinoma cell lines (SW620 and PC/JW), levels of apoptosis induced by
EB1089 were similar or greater than those induced by 1alpha,25-dihydroxyvitamin D3. Although the
carcinoma cell line HT29 was relatively resistant to apoptosis induced by
EB1089 compared with 1alpha,25-dihydroxyvitamin D3,
EB1089 markedly inhibited cell yields. These observations offer promise for the clinical use of
EB1089. To determine whether the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 and
EB1089 was potentially 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 population. A link between the induction of differentiation and apoptosis by 1alpha,25-dihydroxyvitamin D3 and
EB1089 is suggested by the occurrence of apoptosis subsequent to the induction of differentiation. To investigate the molecular pathway to apoptosis induction, members of the Bcl-2 family of
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 not essential for apoptosis. Levels of the proapoptotic
protein Bak, however, were consistently increased in all of the five cell lines in association with apoptosis induced by either agent. The results implicate
Bak protein in the induction of apoptosis by 1alpha,25-dihydroxyvitamin D3 or its analogue
EB1089. The ability of
EB1089 to induce apoptosis in
colorectal carcinoma cells suggests that this or other
vitamin D analogues may prove clinically effective for the treatment of
colorectal cancer. Furthermore, the fact that it induces cell cycle arrest and apoptosis in the premalignant
adenoma cells may suggest an application in
colorectal cancer chemoprevention.