Novel vitamin D₃ analog, 21-(3-methyl-3-hydroxy-butyl)-19-nor D₃, that modulates cell growth, differentiation, apoptosis, cell cycle, and induction of PTEN in leukemic cells

The present chemotherapy of cancer uses agents that are usually toxic to normal cells. On the other hand, induction of cellular differentiation may supplement the use of cytotoxic drugs in several forms of neoplasia, like the successful use of all-trans-retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia. The physiologically active form of vitamin D₃, 1,25(OH)₂D₃, is a member of the secosteroid hormone family, which controls calcium homeostasis and bone metabolism. 1,25(OH)₂D₃ can induce differentiation and inhibit the growth of a number of malignant cell types, including myeloid leukemia, breast, prostate, colon, skin, and brain. Several studies suggested that growth inhibition by 1,25(OH)₂D₃ may be attributed to inhibition of the G₁ to S transition in the cell cycle, which probably is due at least in part to stimulation of expression of the cyclin-dependent kinase inhibitors (CDKIs), p21^waf1 and p27^kip1 as well as induction of programmed cell death.1-4 In a clinical study, oral administration of 1,25(OH)₂D₃ to preleukemic patients was only partially effective5; calcemic side effects prevented the administration of the dosage of the compound needed to achieve the concentration of 1,25(OH)₂D₃ in vivo, which was known to be necessary from our in vitro studies.5,6Therefore, synthesis of vitamin D₃ analogs with potent antiproliferative and differentiation activity against cancer cells with decreased risk of hypercalcemia has received considerable attention.7-14 

Recently, PTEN/MMAC1/TEP1, a tyrosine phosphatase, was identified and mapped to chromosome 10q23.3.15-17,PTEN gene mutations have been observed in a variety of human cancers including breast, prostate, brain, lymphoma, and leukemia.15,18-23Germline deletion of PTEN in the mouse resulted in early embryonic lethality, and heterozygous mice developed malignant neoplasms.24,25 These findings strongly suggested that PTEN is a candidate tumor suppressor.

In this study, a class of newly synthesized vitamin D₃analogs having 2 identical side chains attached to carbon-20 was analyzed. We focused particularly on the most active analog, which has a deletion of C-19, 21-(3-methyl-3-hydroxy-butyl)-19-nor D₃(Gemini-19-nor). This new vitamin D₃ analog was more potent than 1,25(OH)₂D₃ in mediating growth inhibition, differentiation, apoptosis, G₁/G₀arrest of the cell cycle, and expression of p27^kip1. Furthermore, we observed that this compound induced the expression of PTEN in myeloid leukemic cells as the cells underwent differentiation.

The myeloid leukemia (HL-60), breast cancer (MCF-7), and prostate cancer (LNCaP) cell lines were obtained from American Type Culture Collection (Rockville, MD). The NB4 promyelocytic leukemia cell line was provided by Dr Lanotte (INSERM, Hospital Saint-Louis, Paris, France). HL-60, NB4, and LNCaP were cultured in RPMI 1640 with 10% fetal calf serum (FCS). MCF-7 cells were maintained in Dulbecco modified Eagle media with 10% FCS. All 4 cell lines were maintained in a 37°C incubator containing 5% CO₂. After informed consent, mononuclear cells from normal bone marrow were collected by separation on Ficoll-Hypaque (Pharmacia, Piscataway, NJ) gradients at a density of 1.077, and washed in Iscove modified Dulbecco medium (IMDM) containing 10% FCS.

All vitamin D₃ analogs were synthesized by Milan R. Uskokovic (Hoffmann-La Roche, Nutley, NJ). The analogs are shown in Figure 1. The vitamin D₃compounds were dissolved in absolute ethanol at 10⁻³ M as stock solution, which were stored at −20°C and protected from light. 12-O-Tetradecanoylphorbol 13-acetate (TPA) and ATRA were purchased from Sigma (St Louis, MO).

Cells were cultured in a 2-layer soft agar system for either 7 days (HL-60 and NB4) or 10 days (MCF-7 and LNCaP) as described previously.26 Normal bone marrow cells were cultured for 14 days in methylcellulose medium M3234 (Stem Cell Technology, Vancouver, BC, Canada) containing 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF). MCF-7 and LNCaP cells were trypsinized, washed, counted, and plated into 24-well, flat-bottom plates with a total of 1 × 10³ cells/well in a volume of 400 μL/well. The feeder layer was prepared with agar that had been equilibrated at 42°C. Prior to this step, vitamin D₃compounds were pipetted into the wells. After incubation, the colonies were counted. All experiments were done at least 3 times using triplicate plates per experimental point.

Expression of cell surface antigens was determined by flow cytometry. HL-60 cells were cultured with either 1,25(OH)₂D₃ or Gemini-19-nor (10⁻⁸and 10⁻⁷ M) for 4 days. After twice washing with phosphate-buffered saline (PBS), cells were incubated for 30 minutes with fluorescein isothiocyanate (FITC)-conjugated murine antihuman CD11b or antihuman CD14 antibody (DAKO, Carpinteria, CA). Murine IgG1 antibody (DAKO) was used as negative control. Cells were analyzed by a FACScan (Becton Dickinson, Mountain View, CA). HL-60 cells were assessed for their ability to produce superoxide as measured by reduction of nitroblue tetrazolium (NBT), by morphology as detected on cytospin preparations stained with Diff-Quick Stain Set (Baxter Healthcare, Miami, FL). All experiments were independently done at least 3 times. All data were statistically analyzed by Studentt test.

Cell cycle analysis was performed on HL-60 cells incubated for 4 days with either 1,25(OH)₂D₃ or Gemini-19-nor at either 10⁻⁸ or 10⁻⁷ M. The cells were fixed in chilled methanol overnight before staining with 50 μg/mL propidium iodide (PI), 1 mg/mL RNase, and 0.1% NP40. Analysis was performed immediately after staining using a FACScan (Becton Dickinson) and CELLFit program (Becton Dickinson). All experiments were independently performed at least 3 times. All data were statistically analyzed by Student t test.

To study induction of apoptosis by vitamin D₃analogs, annexin V assay (Annexin V-FITC Apoptosis Detection Kit; Pharmingen, San Diego, CA) was performed according to the manufacturer's instructions. Briefly, cells were harvested after exposure with either 1,25(OH)₂D₃ or Gemini-19-nor (10⁻⁸ and 10⁻⁷ M), washed twice with PBS, incubated with FITC-conjugated annexin V and PI for 15 minutes, and measured by FACScan (Becton Dickinson). All experiments were independently done at least 3 times. All data were statistically analyzed by Student t test.

Cells were washed twice in PBS, suspended in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, 1% NP40, 100 μg/mL phenylmethylsulfonyl fluoride, 2 μg/mL aprotinin, 1μg/mL pepstatin, and 10 μg/mL leupeptin), and placed on ice for 30 minutes. After centrifugation at 15 000g for 20 minutes at 4°C, the supernatant was collected. Protein concentrations were quantitated using the Bio-Rad assay (Bio-Rad Laboratories, Hercules, CA). Whole lysates (40 μg) were resolved by 4% to 15% SDS-polyacrylamide gel, transferred to an immobilon polyvinylidene difuride membrane (Amersham, Arlington Heights, IL) and probed with anti-p27^kip1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-PTEN antibody (Santa Cruz Biotechnology), and anti-GAPDH antibody (Research Diagnostics, Flanders, NJ). The blots were developed using the enhanced chemoluminescence (ECL) kit (Amersham). Band intensity was measured using a densitometer and fold increase in expression as compared to control, untreated cells was calculated.

The myeloid leukemia (HL-60 and NB4), breast cancer (MCF-7), and prostate cancer (LNCaP) cells were cloned in soft agar in the presence of various concentrations of vitamin D₃ analogs. All 4 Gemini vitamin D₃ analogs and 1,25(OH)₂D₃ inhibited clonal growth of all 4 cell lines in a dose-dependent manner (Figure2). The effective dose that inhibited 50% colony formation (ED₅₀) was determined (Table1). Gemini-19-nor was approximately 70-, 40 625-, 23-, and 380-fold more potent than 1,25(OH)₂D₃ in mediating clonal growth inhibition of HL-60, NB4, MCF-7, and LNCaP cells, respectively. Gemini 1-F-25-OH was 3- to 6-fold stronger than 1,25(OH)₂D₃ in suppressing clonal growth of the myeloid leukemia cell lines (HL-60 and NB4), but it had the same potency as 1,25(OH)₂D₃ against MCF-7 and LNCaP cells. Furthermore, the potency of Gemini 5,6-trans and Gemini 3-epi were nearly equivalent to 1,25(OH)₂D₃ for all 4 of the cell lines. Because Gemini-19-nor was found to be the most potent compound, all additional experiments focused on this analog.

Clonal growth of normal bone marrow-committed myeloid stem cells (colony-forming units-granulocyte/macrophage [CFU-GM]) were not inhibited by either 1,25(OH)₂D₃ or Gemini-19-nor over a concentration range of 10⁻¹⁰ M to 10⁻⁸ M (Figure 3). At 10⁻⁷ M, both compounds inhibited by 20% to 40% the clonal growth of normal bone marrow CFU-GM.

The induction of expression of the cell surface antigens CD11b and CD14 occurs on HL-60 cells as they undergo differentiation. The ability of Gemini-19-nor and 1,25(OH)₂D₃ to induce CD11b and CD14 was analyzed using flow cytometry (Figure4). A 4-day exposure of HL-60 cells to either 1,25(OH)₂D₃ (10⁻⁷ M) or Gemini-19-nor (10⁻⁷ M) resulted in both producing nearly 90% and 80% CD11b⁺ and CD14⁺ cells, respectively. At 10⁻⁸ M, Gemini-19-nor and 1,25(OH)₂D₃ induced 90% and 50% CD11b⁺ cells, and 75% and 30% CD14⁺ cells, respectively.

The capacity of HL-60 cells to produce superoxide as measured by the reduction of NBT was another marker of differentiation that was used. Gemini-19-nor was more potent than 1,25(OH)₂D₃with either Gemini-19-nor or 1,25(OH)₂D₃(10⁻⁸ M, 4 days) inducing 70% and 30% NBT⁺cells, respectively (Figure 5).

Several vitamin D₃ analogs and 1,25(OH)₂D₃ have been shown to cause apoptosis of several type of cancer cells.27,28 Gemini-19-nor and 1,25(OH)₂D₃ (10⁻⁸ M, 4 days) induced 20% and 6% of HL-60 cells, respectively, to undergo apoptosis (Figure 6). At a higher concentration (10⁻⁷ M, 4 days), both compounds produced apoptosis of about 20% of HL-60 cells (Figure 6).

The effect of Gemini-19-nor and 1,25(OH)₂D₃ on the cell cycle of the HL-60 cells was determined. A significant accumulation (P < .05) of cells in the G₁/G₀and G₂/M phases of the cell cycle occurred, with a concomitant decrease in the proportion of those in S phase after 4 days of culture with either 1,25(OH)₂D₃(10⁻⁷ M) or Gemini-19-nor (10⁻⁸ and 10⁻⁷ M) (Figure 7).

The cyclin-dependent kinase inhibitor, p27^kip1, may act as a key regulator of G₁/G₀ accumulation induced by vitamin D₃. Both 1,25(OH)₂D₃ and Gemini-19-nor induced expression of p27^kip1 in a dose-dependent manner at 4 days of exposure of HL-60 cells as determined by Western blot analysis (Figure 8A). Consistent with the cell cycle results, Gemini-19-nor strongly induced expression of p27^kip1 at a lower concentration (10⁻⁹ M) than 1,25(OH)₂D₃ (10⁻⁷ M). A time course study of HL-60 cells indicated that Gemini-19-nor (10⁻⁷ M) induced p27^kip1 protein expression by approximately 7-fold at day 2 and 14-fold by day 4 of culture (Figure8B).

Recent studies showed that the tumor suppressor PTEN was mutated or otherwise dysregulated in several types of human tumors. It is involved in the normal regulation of cell growth, the cell cycle, and apoptosis.29,30 We evaluated the effect of vitamin D₃ compounds on protein expression of PTEN in HL-60 cells that have no mutation, deletion, or methylation of this gene.22 The nontreated, control HL-60 cells had a very low level of expression of PTEN as determined by Western blot analysis (Figures 8A,B). Expression markedly increased by 16-fold in HL-60 cells at 4 days of culture with 10⁻⁹ M Gemini-19-nor; under the same culture conditions, the 1,25(OH)₂D₃(10⁻⁹ M) did not induce detectable levels of PTEN (Figure8A). At 10⁻⁷ M, Gemini-19-nor and 1,25(OH)₂D₃ increased the expression of PTEN by about 32-fold and 14-fold, respectively. A time course study showed that Gemini-19-nor enhanced expression of PTEN by 6-fold at 0.5 days and about 36-fold at 3 days of exposure (Figure 8B).

We also examined if other inducers of differentiation of HL-60 cells could up-regulate expression of PTEN. The phorbol diester, TPA, induces macrophage-like differentiation31 and ATRA induces granulocyte-like differentiation of HL-60 cells. Four days of culture with either TPA (10⁻⁹ M) or ATRA (10⁻⁷ M) induced PTEN expression in HL-60 cells (Figure 8C).

A recent study revealed that a vitamin D₃ compound that has 2 side chains on C-20 (Gemini) was more active than 1,25(OH)₂D₃ in its ability to inhibit clonal growth of malignant cells.32 Therefore, we synthesized additional novel Gemini compounds and examined their biologic effects on cancer cells. In this study, we evaluated 4 compounds from the newly synthesized family of Gemini. The Gemini-19-nor, which has 2 side chains on C-20 and the removal of the C-19, was the most potent inhibitor of clonal proliferation of myeloid leukemia, breast, and prostate cancer cells. It was more active than 1,25(OH)₂D₃ in these 3 types of cancers. In particular, the analog showed marked activity with the NB4 acute promyelocyte leukemia cells and was 40 625-fold more potent than 1,25(OH)₂D₃. Therefore, we focused on the activity of this analog compared with 1,25(OH)₂D₃.

Previously, we reported that analogs of 1,25(OH)₂D₃ that had removal of their C-19 moiety (19-nor 1,25D₃ analogs) were active against prostate, breast, and myeloid leukemia cells.12,13,33,34The 19-nor analog with the code name LH (1,25[OH]₂-16-ene-23-yne-26,27-F6-19-nor-D₃) was most potent against cancer cells of breast (ED₅₀: MCF-7, 8 × 10⁻¹⁰ M)12 and prostate (ED₅₀: LNCaP, 1 × 10⁻⁹ M),13and the 1,25S(OH)₂-16,23-diene-26-F₃-19-nor-D₃was most potent against myeloid leukemia cells (ED₅₀: HL-60, 4 × 10⁻¹¹ M; NB4, 5 × 10⁻¹¹M).34 When compared with these 19-nor analogs, Gemini-19-nor was about 100-fold more potent than 1,25S(OH)₂-16,23-diene-26-F₃-19-nor-D₃when studied with NB4 cells, whereas it had either comparable or weaker activity than LH and 1,25S(OH)₂-16,23-diene-26-F₃-19-nor-D₃with HL-60, MCF-7, and LNCaP cells. Therefore, even though study conditions were not completely identical, these data suggest that in some types of leukemia, Gemini-19-nor may be a more potent inhibitor of proliferation than 1,25S(OH)₂-16,23-diene-26-F₃-19-nor-D₃, which was the most active vitamin D₃ analog against myeloid leukemia cells in previous studies. In contrast to the effect on tumor cell lines, Gemini-19-nor (10⁻¹⁰ M to 10⁻⁸ M) did not inhibit the clonal growth of normal human bone marrow committed myeloid stem cells (CFU-GM).

Exposure of HL-60 myeloid leukemia cells to either 1,25(OH)₂D₃ or Gemini-19-nor induced the expression of the cell surface markers, CD11b and CD14. Gemini-19-nor (10⁻⁸ M) induced expression of CD11b and CD14 in about 90% and 75% of cells, whereas the same concentration of 1,25(OH)₂D₃ induced expression on only 50% and 30% of cells, respectively. Similarly, Gemini-19-nor (10⁻⁸ M) induced 70% of HL-60 cells to become NBT⁺, compared to only 30% for the same concentration of 1,25(OH)₂D₃. Therefore, these results suggested that Gemini-19-nor was more potent than 1,25(OH)₂D₃ as an inducer of myeloid differentiation.

Gemini-19-nor also mediated apoptosis. Previously, we reported that vitamin D₃ analogs in concert with a RXR ligand induced apoptosis of myeloid leukemia cells and caused levels of expression of Bcl-2 to decrease suggesting an association between the 2 events.3,4,35 However, other experiments found that a vitamin D₃ analog could induce apoptosis of an HL-60 variant without a reduction of cellular levels of Bcl-2.4Another group reported that vitamin D₃ compounds induced apoptosis via a novel caspase- and p53-independent pathway, and apoptosis was inhibited by forced expression of Bcl-2.36These findings suggested that vitamin D₃ might use several pathways to induce apoptosis.

Previous studies showed that vitamin D₃ analogs caused accumulation at the G₁/G₀ phase of the cell cycle, and this block may be mediated by p21^waf1 and p27^kip1 CDKIs.2,26,37 Gemini-19-nor D₃ also produced G₁- to S-phase block of the cell cycle and induced p27^kip1 expression. These results support the hypothesis that CDKIs mediated at least in part the antiproliferative affects of the vitamin D₃ compounds by induction of a G₁/G₀ accumulation. A block in the G₂/M checkpoint has also been previously observed in HL-60 cells treated with 1,25(OH)₂D₃,38 and Gemini-19-nor was able to more potently induce a G₂/M block compared to 1,25(OH)₂D₃. This effect has been attributed to a decrease in levels of p34(cdc),39a protein kinase which associates with B-type cyclins and controls transition through G₂/M. Therefore Gemini-19-nor may be able to decrease levels of this protein to a greater extent than 1,25(OH)₂D₃.

Recent studies indicated that the candidate tumor suppressor, PTEN, could block the phosphatidylinositol 3′-kinase (PI3K)/Akt signaling pathway, resulting in cell death or inhibition of growth or both.29,30 Activated Akt mediates cell survival by inhibition of mitochondrial release of cytochrome c, inactivation of Forkhead transcription factors (FKHR), and phosphorylation of BAD and caspase-9.40 The overexpression of exogenous PTEN induced apoptosis of malignant cells.41-45 Moreover, a genetic link between the Fas proapoptotic pathway and PTEN was suggested, because Fas-mediated apoptosis was impaired in the germline heterozygous PTEN^+/− murine model.46 In addition, PTEN induced G₁ cell cycle arrest and this was associated with an increased expression of p27^kip1.45,47-53 In the present study, Western blotting analysis demonstrated that PTEN expression was up-regulated by vitamin D₃ compounds in HL-60 cells, and it paralleled the induction of apoptosis, expression of p27^kip1 and G₁ cell cycle arrest. These findings suggest that PTEN might enhance apoptosis and G₁ cell cycle arrest in transformed cells exposed to a vitamin D₃ analog, and it might help explain the multiple pathways of apoptosis mediated by vitamin D₃ compounds.

Several studies indicated that PTEN induced cell differentiation in glioma cells.54,55 Therefore, we analyzed inducers of myeloid differentiation other than vitamin D₃. We choose TPA, a stimulator of the protein kinase C pathway that induces macrophage-like differentiation, and ATRA, which binds the nuclear hormone receptor, retinoic acid receptor and induces granulocytic differentiation. As shown in Figure 6C, TPA and ATRA significantly induced PTEN expression. These observations suggest that PTEN expression is associated with monocytic and granulocytic differentiation. We do not know if this marked increase in PTEN expression is the cause or the effect of terminal differentiation of HL-60 cells. Further studies are required to define the role PTEN plays in this process of myeloid differentiation.

Taken together, the new vitamin D₃ analog Gemini-19-nor D₃ strongly inhibited growth of transformed cells, and produced myeloid differentiation, apoptosis, and G₁/G₀ arrest associated with elevated levels of p27^kip1. Moreover, the vitamin D₃analog induced expression of PTEN. These observations suggest that the anticancer effects of vitamin D₃ might be regulated in part via PTEN. This analog may provide an adjuvant therapy for myeloid leukemia, especially acute promyelocytic leukemia, and may be effective in other types of cancers.