Conceptually new deltanoids (vitamin D analogs) inhibit multistage skin tumorigenesis

Thomas W. Kensler3, Patrick M. Dolan, Stephen J. Gange1, Jae-Kyoo Lee2, Qiang Wang2 and Gary H. Posner2

Department of Environmental Health Sciences and
1 Department of Epidemiology, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, MD 21205 and
2 Department of Chemistry, School of Arts and Sciences, The Johns Hopkins University, Baltimore, MD 21218, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Development of vitamin D analogs (deltanoids) as chemopreventive agents requires separation of desirable antiproliferative and pro-differentiating activities from the undesirable calcemic activity also found in the hormone calcitriol (1{alpha},25-dihydroxyvitamin D3). Therefore, several conceptually new deltanoids were synthesized with modifications to the 1{alpha}- and/or 25-hydroxyl groups, positions traditionally considered essential for stimulating biological responses. In this study, 1ß-hydroxymethyl-3-epi-25-hydroxyvitamin D3, a non-calcemic CH2 homolog of the natural hormone with antiproliferative activity in vitro, was ineffective as an inhibitor of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced induction of ornithine decarboxylase activity in mouse epidermis. However, a hybrid analog incorporating not only the calcemia-ablating 1ß-hydroxymethyl alteration, but potentiating C,D ring 16-unsaturation and side chain 24,24-fluorination and 26,27-homologation was found to be as effective as calcitriol. Several non-calcemic 24- or 25-t-butyl sulfones, some containing side chain fluorination but all lacking the 25-hydroxyl group, were also shown to be active in this assay. Three sulfones and the 1ß-hydroxymethyl hybrid were evaluated as inhibitors of multistage carcinogenesis in mouse skin. Female CD-1 mice were initiated with a single dose of 7,12-dimethylbenz[a]anthracene and then promoted twice weekly for 20 weeks with TPA. Deltanoids were applied topically 30 min before TPA. Unlike calcitriol, none of the atypical deltanoids affected body weight gain in these animals. Minimal effects on urinary calcium excretion were observed following chronic treatment with these analogs. All deltanoids inhibited the incidence and multiplicity of papilloma formation, with the hybrid analog showing the greatest efficacy. With this deltanoid, tumor incidence was significantly reduced by 28% and tumor multiplicity by 63%. These results, coupled with the rich chemical diversity available in side chain sulfur-containing deltanoids, particularly when combined with A ring modifications such as 1ß-hydroxylalkyl groups, provide important new advances in the fundamental understanding of chemical structure–biological activity relationships as well as more potent and safe vitamin D analogs for cancer chemoprevention and other medicinal uses.

Abbreviations: calcitriol, 1{alpha},25-dihydroxyvitamin D3; DMBA, 7,12-dimethylbenz[a]anthracene; ODC, ornithine decarboxylase; TPA, 12-O-tetradecanoylphorbol-13-acetate; VDR, vitamin D receptor.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1{alpha},25-Dihydroxyvitamin D3 (calcitriol, 1) (see Figure 1Go) as a hormone plays an essential role in a host of cellular processes involved in calcium phosphate homeostasis, inhibition of cell growth and stimulation of cell differentiation. Although calcitriol has been described as a potent inhibitor of multistage [7,12-dimethylbenz[a]anthracene (DMBA)->12-O-tetradecanoylphorbol-13-acetate (TPA)] skin carcinogenesis (1,2), it produces severe toxicity with chronic dosing. Thus, in practice, the clinical use of vitamin D3 as a template for effective chemopreventive agents will require the development of analogs (deltanoids) separating desirable antiproliferative and pro-differentiating activities from undesirable calcemic activity (3).



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Fig. 1. Structures of 1{alpha},25-dihydroxyvitamin D3 (calcitriol, 1), 1ß-hydroxymethyl calcitriol (2) and the other synthetic deltanoids (36) used in this study.

 
To this end, structural changes in the C,D ring and side chain regions of calcitriol have been made. Analogs with double and triple bonds in the C/D ring and side chain (4), addition of multiple fluoride groups to the end of the side chain (57), ethylation of the 24 position (8) or placement of the side chain in the 20-epi configuration (9) have all been shown to have enhanced antiproliferative behavior and, in some instances, chemopreventive activity. Most notably, 1{alpha},25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24-5531) effectively prevents experimental carcinogenesis in the breast (7), prostate (10) and colon (11) of the rat. These modifications are thought to enhance the bioavailability of the analogs by reducing catabolism and altering plasma protein binding (12) and by influencing their interactions with the nuclear vitamin D receptor (VDR). Analogs can prolong the half-life of the VDR or may induce novel VDR conformations, which may either allow more efficient interactions with vitamin D3 response elements and/or expand the array of vitamin D3 response elements that can be activated (1315). In contrast, the A ring of calcitriol appears to be critical for its calcium regulating activity and modifications in this structure diminish the toxic hypercalcemia of vitamin D metabolites and analogs (16,18).

In order for analogs of calcitriol to stimulate strong biological responses in animals, conventional wisdom has required the presence of both the 1{alpha}- and the 25-hydroxyl groups. These two structurally remote hydroxyl groups have been considered essential for effective binding of the seco-steroid ligand to its nuclear VDR, thereby initiating transcriptional and translational events that mediate the observed diverse physiological responses. Even such a small structural change in the large natural hormone as inverting the stereochemistry of the 1-hydroxyl group or replacing the 1ß-H atom by a 1ß-methyl group has been observed to produce an analog with substantially diminished potency (16). However, we reported that 1ß-hydroxymethyl-3-epi-25-hydroxyvitamin D3 (2), a CH2 homolog of the natural hormone, has strong antiproliferative activity in murine keratinocytes in vitro while being devoid of calcemic effects in rats in vivo (19). Subsequently, a more potent antiproliferative but non-calcemic hybrid analog 3 was prepared (20), incorporating not only the calcemia-ablating 1ß-hydroxymethyl alteration but also the potentiating C,D ring 16-unsaturation and side chain 24,24-difluorination and 26,27-homologation. Other biologically active analogs with modifications at the 1 position have been prepared (21,22). Very recently, we reported that a 25-hydroxyl group is not always necessary for high potency; 24- and 25-t-butylsulfones 46 with natural A ring substituents and stereochemistry are not only potently antiproliferative in murine keratinocytes and malignant melanoma cells in vitro and potent inducers of transcriptional events, but are non-calcemic in rats in vivo (23). Thus, the goal of the studies reported here was to examine whether the rationally designed hybrid analog 3 and the sulfone analogs 46 were efficacious in vivo at preventing tumor promotion in the skin of mice without compromising their normal growth.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Female CD-1 mice (7–9 weeks of age) were obtained from Charles River (Wilmington, MA). Backs of mice were shaved with surgical clippers 2 days prior to experimental use and only those mice in the resting phase of the hair growth cycle were used. All chemicals were applied topically to the shaved area in 0.1 ml acetone. For the assay of ornithine decarboxylase (ODC), mice received topical application of acetone or deltanoid (1 µg) 30 min before treatment with 4 nmol TPA. Deltanoids 2, 3, 4, 5 and 6 were synthesized as described previously (19,20,23) while calcitriol 1 was obtained from Solvay Duphar (Weesp, The Netherlands). Five hours later, mice were asphyxiated with CO and killed by cervical dislocation. Epidermis was isolated by brief heat treatment and ODC activity was assayed in the 12 000 g supernatant of epidermal extracts by measuring the release of 14CO2 from L-[14C]ornithine hydrochloride using an Eppendorf microvessel assay system (24). Assays were performed in triplicate on supernatants prepared from pools of two mice. Protein content was determined as described by Bradford (25), using bovine serum albumin as standard. For tumor induction experiments, mice were initiated with 0.2 µmol DMBA and, beginning 10 days later, were promoted twice weekly with 4 nmol TPA for 20 weeks. Vehicle or deltanoids (1 or 3 µg) were applied 30 min prior to each application of TPA. Animals were weighed weekly and the incidence of tumors was observed and recorded weekly. Responses are presented as the average number of papillomas per mouse and the percent of surviving mice with papillomas.

To determine urinary calcium levels, mice were housed in groups of five in glass metabolism cages during week 20 of promotion for 3 consecutive days after the final treatment with TPA and vehicle or deltanoid. Food and water were provided ad libitum. Daily urine samples, which were collected on ice, were centrifuged at 650 g for 10 min, adjusted to pH 6.0 as necessary and assayed for calcium content spectrophotometrically at 575 nm using reagents and standards from Sigma calcium kit no. 587.

Comparisons of body weights and urinary Ca2+ measurements made at 20 weeks were evaluated using Student's t-test. The effect of deltanoids on the occurrence of tumors was evaluated using three different approaches. First, the incidence of any tumor occurrence in mice was compared using Fisher's exact test. Second, tumor multiplicity was compared using log-linear (Poisson regression) models for comparing the total tumor incident rates for each group. Lastly, the time from exposure to the development of tumors was compared using a modification of the Cox proportional hazards model (26), using methods available in Splus v.5.1 (MathSoft Inc., Seattle, WA). Specifically, the time to the development of each tumor was analyzed, censoring mice that did not develop tumors by the end of the experiment, while adjusting for the correlation between multiple tumors within the same mouse. Similarly to the Cox model for non-correlated data, the model parameters can be interpreted as relative risks for comparing each of the treatment groups to the control group.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The induction of ODC activity is a prominent event after exposure to epidermal tumor promoters (27) and the quantification of ODC activity offers a useful first approach to the identification and characterization of potential antagonists of the tumor promotion process. Phorbol ester-mediated induction of ODC in skin can be inhibited by many classes of chemopreventive agents, including calcitriol (28). As shown in Table IGo, pretreatment with either calcitriol or any of deltanoids 36 engendered an ~40% reduction in TPA-induced activity. In contrast, the 1ß-hydroxymethyl analog 2 was inactive. No significant differences in inhibitory effect were seen between any of the effective analogs and calcitriol when tested at the single 1 µg topical dose.


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Table I. Effect of calcitriol and deltanoids 26 on phorbol ester-stimulated ODC induction in mouse skin
 
In a preliminary study (experiment 1) it was observed that twice weekly topical administration of 1 µg calcitriol to female CD-1 mice led to a statistically significant depression of body weight gain within 4 weeks (data not shown) and mortality in some of these animals shortly thereafter, leading to early termination of that arm of the experiment. In contrast, as shown in Table IIGo, twice weekly topical treatment with 1 µg of hybrid deltanoid 3 had no inhibitory effect on growth rates when compared with solvent-treated controls and, in fact, slightly enhanced body weight gain. In a follow-up study (experiment 2), twice weekly treatment of mice for 20 weeks with 3 µg of deltanoids 36 had no effect on weight gain or maintenance of body weight. As indicated in Table IIGo, terminal body weights were within 5% of control values in all treatment groups.


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Table II. Effect of deltanoids 36 on body weight, urinary calcium excretion and final incidence, multiplicity and relative risk of papillomas
 
As shown in Table IIGo, application of 1 µg of deltanoid 3 30 min prior to each application (twice weekly for 20 weeks) of 4 nmol TPA to the skin of mice initiated with DMBA resulted in a 24% reduction in tumor incidence and a 58% reduction in the number of papillomas per mouse compared with those initiated mice that received vehicle and TPA alone. A follow-up study using a higher dose of 3 (3 µg) yielded modestly better protective effects against tumor incidence and multiplicity. As shown in Figure 2Go and Table IIGo, all of the deltanoids tested (36) significantly reduced the multiplicity of tumors per mouse when applied at a dose of 3 µg (P < 0.001). Deltanoid 3 was significantly more effective (P < 0.001) than 46 at reducing tumor multiplicity. Comparisons of the time for development of papillomas using a modified Cox proportional hazards model indicated that deltanoids 35 significantly delayed tumor development (P < 0.03) while the efficacy of deltanoid 6 was of borderline significance (P < 0.066).



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Fig. 2. Time course of the effects of topical application of deltanoids 36 on the incidence and multiplicity of papillomas induced by an initiation–promotion regimen in mouse skin.

 
Rates of excretion of calcium in the urine is a sensitive monitor for the hypercalcemic effects of calcitriol and its calcemic analogs. As presented in Table IIGo, 20 weeks of twice weekly application of 3 µg of deltanoids 36 had minor effects on urinary levels of calcium during the terminal week of experiment 2. Deltanoid 3 actually reduced urinary calcium excretion while 6 produced a 2-fold increase. Deltanoids 4 and 5 had no effect compared with the control. These results are consistent with our earlier reports that, unlike calcitriol, deltanoids 26 are devoid of calcemic activity in F344 rats (19,20,23) when administered at daily doses up to 10 µg per os.

Over the past several years we have developed a series of highly atypical deltanoids harboring novel structural modifications to the 1{alpha}-hydroxy or/and the 25-hydroxy positions that exhibit strong antiproliferative effects against cultured murine epidermal cells but which do not result in hypercalcemia when administered in vivo to rats (1923). In this report we demonstrate that several of these conceptually new deltanoids are effective chemopreventive agents in a murine initiation–promotion model of skin carcinogenesis, a model in which the efficacy of calcitriol has been previously established (1,2). While the mechanisms for the chemopreventive actions of calcitriol and its analogs remain largely unknown, it appears that genomic effects associated with altered expression of TGF-ß1 and TGF-ß2 (29), epidermal growth factor receptors (30) and cell cycle arrest in G1 (18) may be critical to their actions.

Structural modifications to the 1{alpha}-hydroxy (deltanoids 2 and 3) or/and the 25-hydroxy (deltanoids 36) positions have been introduced into calcitriol. Although an active antiproliferative agent in vitro (19), deltanoid 2 (which only has modifications in the A ring) did not inhibit TPA-induced ODC induction in vivo, unlike the other four analogs tested, which are all modified on the side chain (Table IGo). Interestingly though, deltanoid 3, which is modified at both the A ring and side chain, was the most effective inhibitor of TPA-mediated ODC induction and tumor promotion in this model. Thus, modifications at the 25-hydroxy position are consonant with chemopreventive activity and are apparently facilitated with companion modification of the A ring. The dichotomy between the actions of deltanoids 2 and 3 may be related to their interactions with the VDR, which has been assayed using cells transfected with a plasmid containing the vitamin D response element attached to a growth hormone reporter gene (20). Although the single region-modified 1ß-hydroxymethyl analog 2 with the natural side chain has only weak transcriptional activity in cells in vitro, the corresponding double region-modified hybrid analog 3 with 16-ene-24,24-difluoro structural changes is even more transcriptionally potent (ED50 5x10–11 M) than 1 (ED50 3x10–10 M) (20). Also, the in vitro growth inhibitory potency of hybrid analog 3 in murine keratinocytes and in B16 malignant melanoma cells matches or exceeds that of calcitriol, whereas the growth inhibitory potency of deltanoid 2 is much lower (20). Despite these sharp differences between 1ß-hydroxymethyl analog 2 and the corresponding hybrid analog 3, both bind only weakly to the VDR, relative to the strong binding of 1. An explanation for such high transcriptional and growth potencies despite such low VDR binding has centered on the different degrees to which such deltanoids use the transcription activation function 2 domain of the VDR to induce a conformational change and enhance the stability of VDR–ligand complexes (15). Despite the absence in hybrid analog 3 of the natural 1{alpha}-hydroxy group, we show here in vivo that this non-calcemic deltanoid effectively inhibits multistage skin carcinogenesis in mice without compromising their normal growth.

The presence of the natural A ring with its 1{alpha}-hydroxy group notwithstanding, the single region-modified deltanoids 46 are chemically and biologically atypical. Chemically, in contrast to Ro24-5531, an established chemopreventive deltanoid that has both the natural 1{alpha}-hydroxy and 25-hydroxy groups, deltanoids 46 lack a side chain terminal hydroxy group thought to be crucial for binding to the VDR. We propose that side chain sulfones 46 represent the first members of a conceptually new class of deltanoids in which binding to the VDR occurs with the terminal sulfone acting as a hydrogen bond acceptor, complementary to the hydrogen bond donor function of the terminal 25-hydroxy group of the natural hormone. Competitive binding affinities for these three deltanoids to human VDR (relative to 100% binding for 1) are as follows: 4, 21%; 5, 7.9%; 6, 4% (23). Despite such modest binding affinities to the VDR, deltanoids 4 and 5, for example, are comparable with 1 in transcriptional potency at 1 nM concentration in ROS 17/2.8 cells (23). Furthermore, deltanoids 46 are comparable with 1 in growth inhibitory potency at low nanomolar concentrations in murine keratinocytes in vitro (23). As shown here in vivo, these single region-modified deltanoids 46 can also effectively inhibit multistage skin carcinogenesis in mice without compromising their normal growth. There was no significant difference between the efficacy of 24-fluorinated deltanoids 5 and 6 and that of non-fluorinated deltanoid 4.

In a separate study, we have observed that an unfluorinated 25-t-butylsulfone deltanoid homologous with (one CH2 larger than) non-calcemic unfluorinated 24-t-bulylsulfone analog 4 is, unfortunately, comparable with calcitriol in terms of undesirable calcemic effect in rats. We have observed also that an unfluorinated version of deltanoid 6, although potently antiproliferative in murine keratinocyte and in B16 malignant melanoma cells, is moderately calcemic in rats (unpublished observations). Coupled with the slight hypercalcemic effect of 6 seen in chronically treated mice (Table IIGo), it appears that the hybrid deltanoids containing both A ring and side chain modifications are preferable for high growth inhibitory efficacy and also high safety (i.e. non-calcemic properties).

Chemically, the side chain sulfone groups in deltanoids 36 offer multiple reaction paths for introducing other structural appendages and/or other functional groups. Also, chemically related 24-fluorinated sulfide and sulfoxide deltanoids can be prepared as well as 24-fluorinated sulfonamides and sulfonate esters. Thus, the rich chemical diversity in such side chain sulfur-containing deltanoids is likely to produce important new advances in the fundamental understanding of chemical structure–biological activity relationships as well as possibly more potent and safe vitamin D3 analogs for cancer chemoprevention and other medical uses.


    Notes
 
3 To whom correspondence should be addressed Email: tkensler{at}jhsph.edu Back


    Acknowledgments
 
This work was supported in part by NIH grant CA44530.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received January 11, 2000; revised March 6, 2000; accepted March 15, 2000.