Marked Effect of the Combination of a Novel Vitamin D3 Analog, KH1060, and 9-cis-RA on Inhibition of Clonal Growth, Decreases of bcl-2 Level and Induction of Apoptosis in HL-60 Cells

All-trans retinoic acid (RA) is the first highly effective differentiation-inducing agent for remission induction in patients with APL, but remissions are short-lived because the treatment fails to eliminate the malignant clone and clinical resistance develops rapidly. Another retinoic acid compound, 9-cis-RA, unlike alltrans-RA—which binds only retinoic acid receptors (RARs)—is a high affinity ligand for both RARs and RXRs. To prevent or overcome resistance and to eliminate the malignant clone, in analogy with chemotherapy, combinations of new potent differentiation-inducing drugs working through different receptors and signal pathways may be useful. The active form of vitamin D3 [1,25 dihydroxyvitamin D3 (1, 25D)] is an inhibitor of proliferation and effector of differentiation of myeloid leukemic cells to monocytes. The 20-epi-22oxa-24a, 26a, 27a-tri-homo-1α,25(OH)2D3 (KH 1060) belongs to the family of 20-epi-vitamin D3 analogs and is considerably more potent in vitro than 1,25D as a regulator of growth of the HL-60 cell line. The aim of this study was to evaluate the affect of the combination of KH 1060 with 9-cis-RA on proliferation of the human promyelocytic leukemia cell line HL-60. The 9-cis-RA (10-8M) produced a 30% inhibition of clonal proliferation of HL-60, and KH 1060 at 10-9 M resulted in 50% inhibition of clonal proliferation of the leukemic cells. No colonies were detectable after incubation with 10-7 MKH 1060. The combination of 9-cis-RA (10-8M) with KH 1060 (10-9M) produced a 95% inhibition of clonal growth. When the HL-60 clonogenic cells were cultured in liquid medium with either 10-7 M of KH 1060 or 9-cis-RA for 3 days, washed and plated in soft agar, KH 1060 and 9-cis-RA inhibited 54% and 30% of the HL-60 cells, respectively. In constrast, when the cells were cultured in liquid culture with 10-7 M KH 1060 and 9-cis-RA together for 3 days, washed and plated in soft agar, no colonies were detectable. In order to gain insight into the remarkable antileukemic effect of the combination of these analogs, apoptosis and expression of bcl-2 were examined. After 3 days of culture of HL-60 cells with the combination of KH 1060 (10-7 M) and 9-cis-RA (107 M), apoptosis was induced in 62% of cells, as detected by measurement of morphological changes. Also after 3 days, bcl-2 protein, as analyzed by immuno-histochemistry, became nearly undetectable when cells were cultured with both KH 1060 and 9-cis-RA (10-7 M). When cultured over the same period with either KH 1060 or 9-cis-RA (107 M), 26% and 36% of cells expressed bcl-2, respectively, and 100% of wild-type HL-60 expressed bcl-2. In summary, our data demonstrate that the combination of KH 1060 and 9-cis-RA ireversibly and synergistically inhibited clonal growth and induced apoptosis of HL-60 cells concomitantly with a very marked decrease expression of bcl-2.