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[Cancer Research 59, 4023-4029, August 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 4023-4029, August 15, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

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

Jun-ichi Hisatake2, Tetsuya Kubota, Yasuko Hisatake, Milan Uskokovic, Shigeru Tomoyasu and H. Phillip Koeffler

Division of Hematology/Oncology, Cedars-Sinai Research Institute, UCLA School of Medicine, Los Angeles, California 90048 [J-i. H., T. K., Y. H., H. P. K.]; Hoffmann LaRoche, Inc., Nutley, New Jersey 07110 [M. U.]; and Department of Hematology, Showa University School of Medicine, Tokyo, Japan [S. T.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is the physiologically active form of vitamin D3 that inhibits proliferation and induces differentiation of a variety of malignant cells. We evaluated a newly synthesized vitamin D3 analogue [1,25(OH)2-16-ene-5,6-trans-D3 (Ro 25-4020)] that has a novel 5,6-trans motif. Dose-response studies showed that 1,25(OH)2-16-ene-5,6-trans-D3 had 10–100-fold greater antiproliferative activities than 1,25(OH)2D3 when measuring clonal growth of breast (MCF-7) and prostate (LNCaP) cancer cell lines as well as a myeloid leukemia cell line (HL-60). Because the chief toxicity of vitamin D3 is hypercalcemia, we examined the calcemic activity of 1,25(OH)2-16-ene-5,6-trans-D3 in mice. Remarkably, 1,25(OH)2-16-ene-5,6-trans-D3 was at least 40-fold less calcemic as compared with 1,25(OH)2D3 and 1,25(OH)2-16-ene-D3 (Ro 24-2637). To explore the mechanism by which the 1,25(OH)2-16-ene-5,6-trans-D3 analogue mediated its antiproliferative activity, several studies were performed. Pulse-exposure studies showed that a 4-day pulse exposure to 1,25(OH)2-16-ene-5,6-trans-D3 (10-7 M) in liquid culture was adequate to achieve a 40% inhibition of MCF-7 clonal growth in the absence of the analogue, suggesting that the growth inhibition mediated by 1,25(OH)2-16-ene-5,6-trans-D3 was at least in part irreversible. Cell cycle studies showed that 1,25(OH)2-16-ene-5,6-trans-D3 increased the proportion of MCF-7 cells in the G0-G1 phase and decreased those in the S phase. Furthermore, 1,25(OH)2-16-ene-5,6-trans-D3 induced an elevated expression of the cyclin-dependent kinase inhibitors, p21waf1 and p27kip1. In addition, 1,25(OH)2-16-ene-5,6-trans-D3 almost completely inhibited telomerase activity, as measured by telomeric repeat amplification protocol assay and human telomerase reverse transcriptase mRNA. For each of the growth-related parameters that were examined, the vitamin D3 analogue was more active than 1,25(OH)2D3. In contrast, 1,25(OH)2D3 was more calcemic than 1,25(OH)2-16-ene-5,6-trans-D3. In summary, 1,25(OH)2-16-ene-5,6-trans-D3, having a novel 5,6-trans motif, strongly inhibited clonal proliferation and reduced telomerase activity with low calcemic activity, suggesting further testing in in vivo cancer models. This analogue may gain a therapeutic niche for selected malignancies.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 p21waf1 and p27kip1 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.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
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.

Vitamin D3 Compounds.
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. 1Citation . 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.



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  		Fig. 1. Chemical structures of vitamin D3 compounds used in this study.

 
Soft Agar Colony Assay.
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 x 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.

Serum Calcium Levels in Vivo.
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.

Pulse-Exposure Experiments.
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 by Flow Cytometry.
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.

Western Blot Analysis.
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 x 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-p27kip1 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-p21waf1 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).

Telomerase Activity.
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.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Vitamin D3 Analogues on Clonogenic Growth of Prostate, Breast, and Myeloid Leukemic Cells.
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)Citation . 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)Citation . 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 x 10-11 M, 2.4 x 10-11 M, and 1.9 x 10-12 M for LNCaP, MCF-7, and HL-60 cells, respectively (Table 1)Citation . The ED50s of 1,25(OH)2-16-ene-5,6-trans-D3 was 1.4 x 10-9 M for LNCaP cells, 4.3 x 10-9 M for MCF-7 cells, and 3.0 x 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.



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  		Fig. 2. Dose-response effects of vitamin D3 compounds on clonal proliferation of HL-60, LNCaP, and MCF-7 cells. Results are expressed as a mean percentage of control plates containing no vitamin D3 analogues. Each point represents a mean of three independent experiments with triplicate dishes. Bars, SD.

 

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  		Table 1 Inhibition of clonal proliferation of tumor cells by vitamin D3 analogues

 
Serum Calcium Levels in Vivo.
Because hypercalcemia is a major toxicity of vitamin D3 compounds, we compared their calcemic effects (Fig. 3)Citation . 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.



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  		Fig. 3. The effect of vitamin D3 analogues on serum calcium in mice. {square}, control; {triangleup}, 1,25(OH)2D3 (0.1 µg); {circ}, 1,25(OH)2-5,6-trans-D3 (0.1 µg); •, 1,25(OH)2-16-ene-D3 (0.1 µg); {blacktriangleup}, 1,25(OH)2-16-ene-5,6-trans-D3 (6.0 µg); {blacksquare}, (4.0 µg); x, (2.0 µg); {blacktriangledown}, (1.0 µg); {triangledown}, (0.5 µg); {diamond}, (0.1 µg). Each data point represents the mean; bars, SD. If the SD was <0.2 mg/dl, it does not appear on the graph. The compounds were delivered i.p. on Monday, Wednesday, and Friday.

 
Pulse-Exposure Experiments.
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)Citation . 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.



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  		Fig. 4. Effect of pulse-exposure of vitamin D3 compounds on the clonal growth of MCF-7 cells. MCF-7 cells were exposed to 10-7 M of either 1,25(OH)2D3 or 1,25(OH)2-16-ene-5,6-trans-D3 for the indicated duration, washed thoroughly, and plated in the absence of vitamin D3 compound in soft agar; colonies were enumerated after 14 days of culture. The results are expressed as a mean percentage of colonies in control plates containing cells not exposed to vitamin D3 analogues. Bars, SD.

 
Analysis of the Cell Cycle and Expression of p21waf1 and p27kip1.
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. 5Citation ).



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  		Fig. 5. Cell cycle analysis of MCF-7 cells. Columns, means of three independent experiments; bars, SD. *, P < 0.05 as determined by Student’s t test for difference compared with the control group.

 
The CDKIs known as p21waf1 and p27kip1 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 p21waf1 and p27kip1, as determined by Western blot analysis (Fig. 6A)Citation . Exposure for 1 day to 1,25(OH)2-16-ene-5,6-trans-D3 (10-7 M) increased levels of p21waf1 and p27kip1 by about 3.2–3.5-fold, whereas culture with 1,25(OH)2D3 (10-7 M; 1 day) increased expression of p21waf1 and p27kip1 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 p21waf1 and p27kip1 about 2.8- and 3.4-fold, respectively; and 1,25(OH)2D3 increased expression of p21waf1 and p27kip1 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 p27kip1 in HL-60 cells in a dose-dependent manner (Fig. 6B)Citation .



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  		Fig. 6. Western blot analysis of CDKIs in cell lines cultured with either 1,25(OH)2D3 or 1,25(OH)2-16-ene-5,6-trans-D3. A, time course study of p27kip1 and p21waf1 expression in MCF-7 breast cancer cells. Cells were either untreated (Control) or cultured with either 1,25(OH)2D3 (10-7 M) or 1,25(OH)2-16-ene-5,6-trans-D3 (10-7 M) for 1, 2, and 3 days. Actin was analyzed as a loading control. B, dose-dependent study of p27kip1 expression in HL-60 myeloid leukemic cells. Cells were either untreated (Control) or cultured with 10-9, 10-8, and 10-7 M of either 1,25(OH)2D3 or 1,25(OH)2-16-ene-5,6-trans-D3 for 4 days. Actin was analyzed as a loading control. Results are expressed as fold increase in expression as compared with untreated cells. The band intensity was measured using a densitometer.

 
Telomerase Activity.
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)Citation .



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  		Fig. 7. Telomerase activity was reduced in HL-60 cells cultured with vitamin D3 compounds. Cells were cultured with 10-9, 10-8, and 10-7 M of either 1,25(OH)2D3 or 1,25(OH)2-16-ene-5,6-trans-D3 for 4 days. Results were compared with cells not cultured with vitamin D3 compound (Control, left two lanes). Cell extracts (0.5 µg of protein) were used for TRAP assay either with (+) or without (-) heat inactivation; heat inactivation provides a control. IC, internal control PCR product.

 
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)Citation . 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.



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  		Fig. 8. hTERT expression in HL-60 cells treated with either 1,25(OH)2-16-ene-5,6-trans-D3 or 1,25(OH)2D3 (10-9, 10-8, 10-7 M; 4 days). Control shows untreated cells. Upper panel, hTERT mRNA expression measured by RT-PCR. Lower panel, GAPDH mRNA was measured as a control.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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 x 10-11 M; MCF-7, 4.3 x 10-9 M; LNCaP, 1.4 x 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)Citation . 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.


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  		Table 2 Relative therapeutic potency

 
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, p21waf1 and p27kip1, 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 p27kip1 and p21waf1 in several cancer cell lines (26 , 27) . In this study, 1,25(OH)2-16-ene-5,6-trans-D3 markedly enhanced expression of p27kip1 and p21waf1 in MCF-7 cells and p27kip1 expression in HL-60 cells. These results were consistent with the hypothesis that the p27kip1 and p21waf1 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.


   ACKNOWLEDGMENTS
 
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.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by United States Defense and NIH grants and by the Lymphoma Foundation, Parker Hughes Trust, CaP Cure, Aaron Eschman Trust, and the C. and H. Koeffler Fund. Back

2 To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, B-215, 8700 Beverly Boulevard, Los Angeles, CA 90048. Phone: (310) 855-4609; Fax: (310) 659-9741. Back

3 The abbreviations used are: 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; Ro 25-4020, 1,25(OH)2-16-ene-5,6-trans-D3; CDKI, cyclin-dependent kinase inhibitor; TRAP, telomeric repeat amplification protocol; hTERT, human telomerase reverse transcriptase; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received 1/ 6/99. Accepted 6/14/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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