20-EPI-VITAMIN-D-3 ANALOGS - A NOVEL CLASS OF POTENT INHIBITORS OF PROLIFERATION AND INDUCERS OF DIFFERENTIATION OF HUMAN BREAST-CANCER CELL-LINES

We have studied the in vitro biological activities and mechanism of ac tion of 1,25-dihydroxyvitamin D-3 (1,25D(3)) and four potent 1,25D(3) analogues [20-epi-22oxa-24a,26a,27a-tri-homo-1,25(OH)(2)D-3 (KH 1060); 20-epi-1,25(OH)(2)D-3; 1,25(OH)(2)-16ene-D-3; and 1,25(OH)(2)-16ene-2 3yne-D-3] on proliferation and differentiation of estrogen receptor-ne gative (MDA-MB-436, BT-20, SK-BR-3, and MDA-MB-231), estrogen receptor -weakly positive (BT474), and estrogen receptor-positive (MCF-7) breas t cancer cell lines. Dose-response studies showed that KH 1060 was the most potent analogue, because it was able to induce differentiation i n ah seven breast cancer cell lines (measured by lipid staining) and t o suppress more than 50% clonal proliferation (ED(50)) at 10(-10) M in all cell lines, except MDA-MB-436 and BT-20. To explore how these com pounds mediated antiproliferative actions, their effects on the cell c ycle, on expression of bcl-2 and p53, and on apoptosis were assessed. Five of six cell lines have a mutant p53 gene, whereas MCF-7 has wild- type p53. Immunohistochemical staining showed that the p53 protein was predominantly localized in the nucleus in each of the breast cancer c ell lines except for MCF-7, which expressed the protein predominantly in the cytoplasm. After incubation with KB 1060 (3 days; 10(-7) M), ex pression of bcl-2 protein as determined by immunohistochemical localiz ation was markedly decreased in BT-474, MCF-7, and MDA-IMB-231; these same cells were profoundly inhibited in their clonal proliferation and arrested in the G(0)G(1) phase of the cell cycle when cultured with K H 1060. In contrast, BT-20 and MDA-MB-436 cells that were refractory t o the antiproliferative effect of KH 1060 (ED(50)<10(-6) M) had no dow n-regulation of their bcl-2 expression and no cell cycle changes after exposure to KH 1060. MCF-7 showed morphological changes and DNA fragm entation, indicative of apoptosis after 48 h incubation with KH 1060 ( 10(-6) M, during which tin;e p53 protein accumulated in the nucleus an d decreased in the cytoplasm. In contrast, no apoptosis was detected i n three other breast lines MDA-MB-231, SK-BR-3, and BT-474) that had a mutated p53. In conclusion, the data indicate that KH 1060 is an extr emely potent 1,25D(3) analogue inducing differentiation of all six bre ast cancer lines and potently inhibiting clonal growth of four of them with concomitant decreased bcl-2 and cell cycle arrest at G(0)G(1). O nly one (MCF-7) of six breast cancer cell lines underwent apoptosis; t hese cells have a wild-type p53 that translocated from the cytoplasm t o the nucleus during culture with KH 1060, probably allowing p53 to be come a functional nuclear transcriptional activator.