A Vitamin D3 Analog Induces a G1-Phase Arrest in CaCo-2 Cells by Inhibiting Cdk2 and Cdk6: Roles of Cyclin E, p21Waf1, and p27Kip1

Abstract

Previous studies by our laboratory have shown that a noncalcemic fluorinated analog of 1alpha,25-dihydroxyvitamin D3, 1alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholcal ciferol (F6-D3), significantly reduced the frequency of colonic adenomas and completely abolished the development of colonic adenocarcinomas in rats treated with azoxymethane. The mechanisms involved in this analog's chemopreventive actions, however, remain unclear. In the present study, we now show that although both 1alpha,25-dihydroxyvitamin D3 and F6-D3 inhibited the proliferation of CaCo-2 cells, a human colonic adenocarcinoma cell line, by increasing their doubling times, only F6-D3 caused an arrest of these cells in the G1 phase of their cell cycle. This arrest was accompanied by an increase in the expression of the cyclin-dependent kinase (cdk) inhibitor proteins, p2Waf1 and p27Kip1, which served to decrease the activity of cyclin-dependent kinase 2 and cyclin-dependent kinase 6, whereas the expression and phosphorylation of pRB were unchanged. In contrast to the increased expression of these cdk inhibitors, the expression of cyclin E was decreased, which further inhibited the activity of cyclin-dependent kinase 2. Collectively, the inhibition of these cyclin-dependent kinases served to arrest the CaCo-2 cells, independent of changes in pRB. Furthermore, antibody neutralization studies suggest that transforming growth factor-beta may mediate the coassociations between cdk2 and p27Kip1 and cyclin E induced by F6-D3. These data indicate that cell cycle arrest may, at least in part, underlie the chemopreventive actions of F6-D3 observed in the azoxymethane model of colon cancer. Furthermore, if the antiproliferative action observed in CaCo-2 cells also occurs in human colonic epithelium, F6-D3 may have chemopreventive potential against human colon cancer, as well.

Transforming growth factor-β1 signaling contributes to Caco-2 cell growth inhibition induced by 1,25(OH)2D3

“…Specifically, we found that this secosteroid causes an increased doubling time (41). In the previous report, however, we did not examine the effects of TGF-␤ antibodies on Caco-2 cell growth inhibition by 1,25(OH) 2 D 3 .…”

“…Our previous studies demonstrated that 1,25(OH) 2 D 3 significantly inhibited Caco-2 cell growth in a dose-dependent manner (10 Ϫ10 -10 Ϫ7 M), with a maximal inhibition at 100 nM (16) and no toxicity up to 300 nM as assessed by lactate dehydrogenase release (41). On the basis of these observations, we chose to study preconfluent Caco-2 cells treated with 1,25(OH) 2 D 3 at 100 nM for these experiments.…”

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

“…Furthermore, Caco-2 cells integrate a complex array of signals during this process. Butyrate and vitamin D 3 are examples of compounds that affect Caco-2 differentiation through overlapping or complementary pathways (43,44). Our results suggest that prostaglandins such as PGE 2 could also be involved in Caco-2 cell differentiation.…”

Antineoplastic effects of 1,25(OH)2D3 and its analogs in breast, prostate and colorectal cancer

“…1,25(OH) 2 D 3 is also able to modulate cellular growth by influencing other important signaling pathways. The transforming growth factor-b (TGF-b) signalization pathway is activated by 1,25(OH) 2 D 3 and contributes to the antiproliferative effects of 1,25(OH) 2 D 3 possibly by mediating coassociations between CDK2, P27 and cyclin E (Scaglione-Sewell et al 2000).…”