5,6-trans-16-ene-Vitamin D₃: A New Class of Potent Inhibitors of Proliferation of Prostate, Breast, and Myeloid Leukemic Cells¹ Free

In the United States, breast and prostate cancers are two of the most prevalent, nonskin malignancies. Improvement in hormonal and cytotoxic therapies has not led to either a major lengthening of remissions or increase in cures in advanced breast cancer. In prostate cancer, blockade of androgen stimulation often leads to a remission; however, a subsequent relapse almost invariably occurs within a few years, resulting in a poorly differentiated, androgen-independent cancer. The present chemotherapy of cancer uses agents that are toxic to normal cells. On the other hand, induction of cellular differentiation may be useful for several forms of neoplasia, like the successful use of all-trans-retinoic acid in the treatment of acute promyelocytic leukemia.

1,25(OH)₂D₃³ is a member of the seco-steroid hormone family, which controls calcium homeostasis. The effects of 1,25(OH)₂D₃ are mediated mainly via interaction with a specific nuclear vitamin D₃ receptor, which heterodimerizes with the retinoic acid receptor (retinoid X receptor; Ref. 1). 1,25(OH)₂D₃ can inhibit the growth and induce differentiation of a variety of types of malignant cells, including breast (2, 3, 4, 5, 6), prostate (7, 8, 9, 10), blood (11, 12, 13, 14), colon (15, 16), skin (17), and brain (18). However, the calcemic side effects of 1,25(OH)₂D₃ have prevented its application as a therapeutic agent (19). Synthesis of analogues of 1,25(OH)₂D₃ with potent antiproliferative and differentiation activity against cancer cells with decreased risk of inducing hypercalcemia have been reported (20, 21, 22, 23, 24, 25).

In the present study, we analyzed 1,25(OH)₂D₃ analogues that have a novel 5,6-trans motif. The results indicated that 1,25(OH)₂-16-ene-5,6-trans-D₃ (Ro 25-4020) was more potent than 1,25(OH)₂D₃ in its ability to inhibit the clonal cell growth of breast (MCF-7) and prostate (LNCaP) cancer cells and myeloid leukemia cells (HL-60) in vitro. Further studies showed that 1,25(OH)₂-16-ene-5,6-trans-D₃ arrested MCF-7 cells in G₀-G₁, which was associated with the rapid and prominent accumulation of the p21^waf1 and p27^kip1 CDKIs. In addition, we showed that 1,25(OH)₂-16-ene-5,6-trans-D₃ markedly inhibited telomerase activity as measured by TRAP assay and hTERT mRNA expression in HL-60 cells.

The breast cancer (MCF-7), prostate cancer (LNCaP), and myeloid leukemia (HL-60) cell lines were obtained from American Type Culture Collection (Rockville, MD). MCF-7 cells were maintained in DMEM with 10% FCS. LNCaP and HL-60 were cultured in RPMI 1640 with 10% FCS. All three cell lines were maintained in a 37°C incubator containing 5% CO₂.

In this study, 10 vitamin D₃ analogues were used: [1,25(OH)₂-16-ene-5,6-trans-D₃; 1,25(OH)₂-5,6-trans-D₃; 1,25(OH)₂-16-ene-D₃; 1,25(OH)₂D₃; 1,25(OH)₂-16,23Z-diene-5,6-trans-D₃; 1,25(OH)₂-16-ene-23-yne-26,27-F₆-5,6-trans-D₃; 1,25(OH)₂-16,23E-diene-26,27-F₆-20-epi-5,6-trans-D₃; 1,25R(OH)₂-16,23E-diene-26-F₃-5,6-trans-D₃; 1,25R,S-(OH)₂-16-ene-23-yne-26-F₃-5,6-trans-D₃; and 25(OH)-16-ene-23-yne-5,6-trans-D₃]. All analogues were synthesized by Hoffmann-La Roche, Inc. The analogues studied in greatest detail are shown in the Fig. 1. The vitamin D₃ compounds were dissolved in absolute ethanol at 10⁻³ m as stock solution, which were stored at −20°C and protected from light. For in vitro use, analogues were diluted in DMEM or RPMI 1640. For in vivo use, analogues were diluted with PBS. An aliquot was used only once.

Cells were cultured in a two-layer soft agar system for either 14 days (MCF-7 and LNCaP) or 10 days (HL-60) as described previously (26). MCF-7 and LNCaP cells were trypsinized. Washed single-cell suspensions of cells were enumerated and plated into 24-well, flat-bottomed 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, compounds were pipetted into wells. After incubation, the colonies were counted. All experiments were done at least three times using triplicate plates per experimental point.

Forty male BALB/c mice at 8–9 weeks of age were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and maintained in pathogen-free conditions and fed a standard laboratory diet. Four mice per group were injected i.p. every other day (except Saturday and Sunday) with either a vitamin D₃ analogue or diluent (100 μl/mouse) for either 3 or 5 weeks. Doses of 1,25(OH)₂-16-ene-5,6-trans-D₃ were 0.1, 0.5, 1.0, 2.0, 4.0, and 6.0 μg/mouse. Doses of 1,25(OH)₂D₃, 1,25(OH)₂-16-ene-D₃, and 1,25(OH)₂-5,6-trans-D₃ were 0.1 μg/mouse based on prior experiments (22, 23). Control mice were injected with 100 μl of PBS. Serum calcium values were measured every week by the quantitative, colorimetric detection assay using the Sigma 587 kit.

The MCF-7 cells were incubated in liquid culture with 10⁻⁷ m of either 1,25(OH)₂D₃ or 1,25(OH)₂-16-ene-5,6-trans-D₃ for various durations. After incubation, these cells were carefully washed twice with PBS, and viable cells were counted and plated into 24-well plates for soft agar colony assay, as described previously (27).

Cell cycle analysis was performed on MCF-7 cells incubated for 4 days with either 1,25(OH)₂D₃ or 1,25(OH)₂-16-ene-5,6-trans-D₃ at 10⁻⁷ m. The cells were fixed in chilled methanol overnight before staining with 50 μg/ml propidium iodide, 1 mg/ml RNase (100 units/ml; Sigma Chemical Co.), and 0.1% NP40 (Sigma Chemical Co., St. Louis, MO). Analysis was performed immediately after staining using a FACScan (Becton Dickinson, Mountain View, CA) and CELLFit program (Becton Dickinson). All experiments were done at least three times independently. All data were statistically analyzed by Student’s t test.

Cells were washed twice in PBS, suspended in lysis buffer [50 mm Tris (pH 8.0), 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, 100 μg/ml phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 1 μg/ml pepstatin, and 10 μg/ml leupeptin], and placed on ice for 30 min. After centrifugation at 15,000 × g for 20 min at 4°C, the supernatant was collected. Protein concentrations were quantitated using the Bio-Rad assay (Bio-Rad Laboratories, Hercules, CA). Whole lysates (15 μg) were resolved by 15% SDS polyacrylamide gel, transferred to an Immobilon polyvinylidene difuride membrane (Millipore Corp., Bedford, MA), and probed with anti-p27^kip1 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-p21^waf1 murine monoclonal antibody (Oncogene Science, Uniondale, NY), and antiactin murine monoclonal antibody (Oncogene). The blots were developed by using the ECL kit (Amersham Corp., Arlington Heights, IL).

To detect the relative telomerase activity, TRAP assays were performed using TRAP-eze telomerase detection kit (Oncor, Gaithersburg, MD) according to the manufacturer’s instruction. For hTERT, total RNAs were isolated from HL-60 cells that were treated with either 1,25(OH)₂D₃ or 1,25(OH)₂-16-ene-5,6-trans-D₃ (10⁻⁹, 10⁻⁸, and 10⁻⁷ m) for 4 days with Trizol (Life Technologies, Grand Island, NY). RT-PCR was performed with 1 μg of total RNA and random hexamer primers using the Superscript Preamplification System (Life Technologies). cDNA was amplified using primers specific for either the hTERT gene or the GAPDH gene, the latter of which was used as a control. The primers used for hTERT were 5′-CGGAAGAGTGTCTGGAGCAA-3′ (sense) and 5′-GGATGAAGCGGAGTCTGGA-3′ (antisense). The thermal cycles were 94°C for 90 s, followed by 33 cycles of 95°C for 20 s, 68°C for 40 s, and 72°C for 30 s. These primers and the PCR conditions were as described previously (28). Primers for the GAPDH gene were 5′-CCATGGAGAAGGCTGGGG-3′ (sense) and 5′-CAAAGTTGTCATGGATGACC-3′ (antisense). Conditions for GAPDH amplifications were: 94°C for 2 min, 26 cycles of 94°C for 30 s, 62°C for 40 s, and 72°C for 60 s, followed by 72°C for 4 min. PCR products were electrophoresed on 1% agarose gel and stained with ethidium bromide.

The LNCaP, MCF-7, and HL-60 cells were cloned in soft agar in the presence of various concentrations of vitamin D₃ analogues at 10⁻¹¹ to 10⁻⁷ m. Dose-response curves were drawn, and the effective dose that inhibited 50% colony formation (ED₅₀) was determined. Initially, 10 analogues were examined (see “Materials and Methods”). The most effective was 1,25(OH)₂-16-ene-5,6-trans-D₃ (Ro 25-4020; data not shown). Thus, additional experiments focused on this analogue. For comparison, 1,25(OH)₂-5,6-trans-D₃, 1,25(OH)₂-16-ene-D₃, and 1,25(OH)₂D₃ were also studied in the additional experiments (Fig. 1). Each of the four vitamin D₃ compounds was effective in inhibition of clonal proliferation of the three cell lines in a dose-dependent manner (Fig. 2). The 1,25(OH)₂-16-ene-D₃ was the most potent. The ED₅₀ of 1,25(OH)₂-16-ene-D₃ was 5.0 × 10⁻¹¹ m, 2.4 × 10⁻¹¹ m, and 1.9 × 10⁻¹² m for LNCaP, MCF-7, and HL-60 cells, respectively (Table 1). The ED₅₀s of 1,25(OH)₂-16-ene-5,6-trans-D₃ was 1.4 × 10⁻⁹ m for LNCaP cells, 4.3 × 10⁻⁹ m for MCF-7 cells, and 3.0 × 10⁻¹¹ m for HL-60 cells, which were about 10–100-fold more potent than 1,25(OH)₂D₃. The potency of 1,25(OH)₂-5,6-trans-D₃ was nearly equivalent to 1,25(OH)₂D₃.

Because hypercalcemia is a major toxicity of vitamin D₃ compounds, we compared their calcemic effects (Fig. 3). The mice that received 0.1 μg of either 1,25(OH)₂D₃ or 1,25(OH)₂-16-ene-D₃ (three times per week) were hypercalcemic, with mean serum calcium levels of approximately 11.6 ± 0.1 and 13.8 ± 0.5 mg/dl (normal, 8.5–10.5 mg/dl) at week 3, respectively. In contrast, mice that received 1,25(OH)₂-16-ene-5,6-trans-D₃ (0.1–2.0 μg/mouse) had almost the same calcium level (7.9–10.4 mg/dl) as the control mice (8.4–9.7 mg/dl). When 6.0 μg/mouse 1,25(OH)₂-16-ene-5,6-trans-D₃ were administered, serum calcium levels increased to a mean 12.9 ± 0.7 and 11.4 ± 0.3 mg/dl at weeks 3 and 5, respectively. When 4.0 μg/mouse were administered, serum calcium levels increased to a mean 11.7 + 0.8 mg/dl at week 3; but at weeks 4 and 5, levels returned to the normal range.

To investigate whether the inhibition of clonogenic proliferation by 1,25(OH)₂-16-ene-5,6-trans-D₃ was reversible, we performed pulse-exposure experiments. The MCF-7 cells were exposed to either 1,25(OH)₂-16-ene-5,6-trans-D₃ or 1,25(OH)₂D₃ for various durations, washed thoroughly, and plated in soft agar; clonal growth was determined on day 14 of culture (Fig. 4). Approximately 40 and 30% of the clonogenic cells were inhibited by 4 days of exposure to 1,25(OH)₂-16-ene-5,6-trans-D₃ and 1,25(OH)₂D₃, respectively, suggesting that these vitamin D₃ compounds were capable of mediating a partial, irreversible inhibition of growth of MCF-7 cells.

To understand better the mechanism by which the vitamin D₃ analogue prevented cell growth, its effect on the cell cycle of the MCF-7 cells was determined. A significant accumulation (P < 0.05) of the number of cells in the G₀-G₁ phase of the cell cycle occurred with a concomitant decrease in the proportion of cells in S phase after culture with either 1,25(OH)₂-16-ene-5,6-trans-D₃ or 1,25(OH)₂D₃ (10⁻⁷ m; 4 days; Fig. 5).

The CDKIs known as p21^waf1 and p27^kip1 are able to inhibit the activity of cyclin kinase and thus slow the progression of the cells through the cell cycle. The control MCF-7 cells constitutively had a moderate level of expression of p21^waf1 and p27^kip1, as determined by Western blot analysis (Fig. 6,A). Exposure for 1 day to 1,25(OH)₂-16-ene-5,6-trans-D₃ (10⁻⁷ m) increased levels of p21^waf1 and p27^kip1 by about 3.2–3.5-fold, whereas culture with 1,25(OH)₂D₃ (10⁻⁷ m; 1 day) increased expression of p21^waf1 and p27^kip1 about 1.6–1.8-fold. By 3 days, 1,25(OH)₂-16-ene-5,6-trans-D₃ (10⁻⁷ m) increased expression of p21^waf1 and p27^kip1 about 2.8- and 3.4-fold, respectively; and 1,25(OH)₂D₃ increased expression of p21^waf1 and p27^kip1 by 4.8- and 3.3-fold, respectively. The 1,25(OH)₂-16-ene-5,6-trans-D₃ and 1,25(OH)₂D₃ also up-regulated expression of p27^kip1 in HL-60 cells in a dose-dependent manner (Fig. 6 B).

Maintenance of telomeres is important for cellular well-being, and progressive telomere shortening limits the replicative capacity of cells (29, 30, 31, 32, 33). Telomerase activity is inhibited by diverse agents including 1,25(OH)₂D₃ during terminal differentiation of leukemia cell lines (34, 35, 36). Therefore, we evaluated the effect 1,25(OH)₂-16-ene-5,6-trans-D₃ and 1,25(OH)₂D₃ (10⁻⁹ to 10⁻⁷ m; 4 days) on telomerase activity using the TRAP assay. Telomerase activity markedly decreased in HL-60 cells cultured with either 10⁻⁹ m 1,25(OH)₂-16-ene-5,6-trans-D₃ or 10⁻⁷ m 1,25(OH)₂D₃ (Fig. 7).

Recent investigations have revealed that hTERT is the catalytic human telomerase subunit, and it plays an important role in the activation of telomerase (37, 38, 39, 40). The effects of vitamin D₃ analogues on hTERT expression in HL-60 cells were evaluated using RT-PCR (Fig. 8). Both 1,25(OH)₂-16-ene-5,6-trans-D₃ and 1,25(OH)₂D₃ inhibited the expression of hTERT mRNA in a dose-dependent manner with almost complete inhibition of expression occurring at 10⁻⁸ m 1,25(OH)₂-16-ene-5,6-trans-D₃ and at 10⁻⁷ m 1,25(OH)₂D₃.

Previously, we showed that the vitamin D₃ analogue having the C-16-ene motif [1,25(OH)₂-16-ene-D₃] was more potent than 1,25(OH)₂D₃ in inhibiting the proliferation of HL-60 cells, but the calcemic activity of this analogue was nearly identical to 1,25(OH)₂D₃ (22). The desaturation of the side chain with the addition of a C-23-triple bond [1,25(OH)₂-16-ene-23-yne-D₃] also was potent in its ability to induce cell differentiation and inhibit leukemic clonal proliferation with low calcemic activity (13, 14, 22). This latter analogue, however, did not have a broad range of activity against breast and prostate cancer cells (27, 41). The addition of six fluorines to the end of the side chain (C-26,27-F₆) markedly enhanced the range and potency of the analogues (24, 25, 26, 27, 41, 42). In particular, 1,25(OH)₂-16-ene-23-yne-19-nor-26,27-F₆D₃ is one of the most potent vitamin D₃ analogues in its ability to inhibit clonal proliferation of breast and prostate cancer cells as well as leukemic cells. But a major toxicity with these fluorine-substituted analogues was their potent induction of hypercalcemia in experimental animals (23, 24). In this study, we evaluated in detail a compound from the newly synthesized family of analogues having a C-5,6-trans motif. The 1,25(OH)₂-16-ene-5,6-trans-D₃ strongly inhibited clonal proliferation of each of the cell lines (ED₅₀: HL-60, 3.0 × 10⁻¹¹ m; MCF-7, 4.3 × 10⁻⁹ m; LNCaP, 1.4 × 10⁻⁹ m). It was approximately 10–100-fold more active than 1,25(OH)₂D₃. In contrast, the 1,25(OH)₂-5,6-trans-D₃ inhibited cell growth almost to the same degree as 1,25(OH)₂D₃. The 1,25(OH)₂-16-ene-D₃, which has a 16-ene motif, was almost 1000-fold more potent than 1,25(OH)₂D₃ in HL-60 cells as described previously (22). It was ∼1000-fold more potent than 1,25(OH)₂D₃ against breast (MCF-7) and prostate (LNCaP) cancer cell lines. These findings show the importance of the 16-ene motif for antiproliferative activity toward cancer cells.

The dose-limiting toxicity of vitamin D₃ compounds is hypercalcemia. The calcium studies in mice highlighted the importance of the 5,6-trans motif. The 1,25(OH)₂-16-ene-5,6-trans-D₃ had very weak calcemic effects, causing no hypercalcemia at week 5 of administration of 4.0 μg, which was given i.p. three times per week. When the dose of the analogue was increased to 6.0 μg, serum calcium levels became elevated at approximately 11 mg/dl on week 5. In contrast, 1,25(OH)₂D₃ and 1,25(OH)₂-16-ene-D₃ at a 40-fold lower dose (0.1 μg/mouse) induced hypercalcemia. Thus, the addition of the 5,6-trans motif to the 16-ene-containing analogue plays an important role in reducing the calcemic activity of the vitamin D₃ analogues.

Although the addition of the 5,6-trans motif decreased the calcemic potential of the 16-ene-analogue, it also decreased its antiproliferative potency against the target cancer cell lines. To compare the ability of the compounds to inhibit clonal growth and to cause hypercalcemia, we calculated the relative therapeutic potency of the compounds (Table 2). When the results were standardized for their potential to cause hypercalcemia, 1,25(OH)₂-16-ene-5,6-trans-D₃ showed a 2.5- and 1.4-fold greater therapeutic index than 1,25(OH)₂-16-ene-D₃ for growth inhibition of leukemic cells (HL-60) and prostate cancer cells (LNCaP), respectively. For the breast cancer cells (MCF-7), however, 1,25(OH)₂-16-ene-5,6-trans-D₃ had a 4.5-fold lower therapeutic potency than 1,25(OH)₂-16-ene-D₃.

We explored the mechanism by which the vitamin D₃ analogues decreased the clonal growth of cancer cells. The vitamin D₃ compounds, including 1,25(OH)₂D₃, increased the number of MCF-7 cells in G₁ and decreased those in S phase. Recently, studies reported that the CDKIs, p21^waf1 and p27^kip1, mediated the G₁ arrest induced by 1,25(OH)₂D₃ in HL-60 cells (43, 44). We have shown previously that vitamin D₃ compounds induced increased expression of p27^kip1 and p21^waf1 in several cancer cell lines (26, 27). In this study, 1,25(OH)₂-16-ene-5,6-trans-D₃ markedly enhanced expression of p27^kip1 and p21^waf1 in MCF-7 cells and p27^kip1 expression in HL-60 cells. These results were consistent with the hypothesis that the p27^kip1 and p21^waf1 mediated at least in part the antiproliferative effects of the vitamin D₃ compounds by producing a G₁-S phase block of the cell lines.

Furthermore, pulse-exposure of MCF-7 breast cancer cells to 1,25(OH)₂-16-ene-5,6-trans-D₃ (10⁻⁷ m; 4 days), followed by extensive washing, plating in soft agar, and enumerating colony formation at 14 days resulted in a 40% inhibition of colony formation. These results showed that 1,25(OH)₂-16-ene-5,6-trans-D₃ inhibited growth of these cancer cells by a mechanism other than one that was merely cytostatic, and only a relatively brief exposure (4 days) was required to cause this growth suppression. This suggests that brief pulse exposures in vivo might suffice for a cancer-suppressive effect.

Telomere length correlates closely with cellular senescence. Cellular senescence appears to be impaired in telomerase-deficient mice (45). Studies have observed that several reagents reduced the telomerase activity during the induction of terminal differentiation of leukemia cell lines (34, 35, 36). In this study, the TRAP assay showed that 1,25(OH)₂-16-ene-5,6-trans-D₃ (10⁻⁸ m; 4 days) almost completely inhibited telomerase activity in HL-60 cells; and within the limits of the TRAP assay, 1,25(OH)₂-16-ene-5,6-trans-D₃ was more potent than 1,25(OH)₂D₃. hTERT is probably the human telomerase catalytic subunit (37, 38, 39, 40), the expression of which significantly correlates with telomerase activity in malignant cell lines and cancer tissue (46, 47). hTERT expression in tumor cells was down-regulated by several inducers of differentiation [retinoic acid (39) or phorbol diester (48)]. We have found that vitamin D₃ compounds caused the down-regulation of hTERT mRNA expression, and this correlated with down-regulation of telomerase activity. These observations suggest that vitamin D₃ compounds inhibit telomerase activity by reducing hTERT mRNA expression. We do not know if this marked decrease in telomerase activity is the cause or the result of CDKI-induced inhibition of growth and terminal differentiation of HL-60 cells.

Taken together, the new vitamin D₃ compound 1,25(OH)₂-16-ene-5,6-trans-D₃ strongly inhibited cell proliferation, caused a G₁-G₀ block in the cell cycle associated with an elevation of expression of several CDKIs, and markedly decreased telomerase activity; and yet, it had extremely low calcemic activity. This analogue should be studied in vivo with several cancer models as a prelude to possible future clinical trials.

We thank Chloe Koeffler and Seth Robinson for enthusiastic help. We also thank Patricia Lin (Flow Cytometry Core Facility, Cedars-Sinai Medical Center, Los Angeles, CA) for generous technical assistance and Kim Burgin for excellent secretarial help.