Introduction

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1,25-Dihydroxyvitamin D₃ (1,25-(OH)₂D₃) plays an important role in control of cellular growth and differentiation of a large number of cell types. Its potent antimitogenic and prodifferentiating properties, which have been demonstrated in normal as well as tumor cells (Studzinski et al., 1993), can, however, not yet be exploited for cancer treatment because of the severe hypercalcemia that would inevitably result from chronic administration of the steroid hormone. For this reason efforts have been undertaken in the past decade or so to design and synthesize analogs of 1,25-(OH)₂D₃ that would lack the hypercalcemic potency of the steroid hormone but nevertheless retain much of its growth inhibitory potential. In this respect, a series of vitamin D compounds bearing structural modifications at metabolically labile sites, viz., in the C and D ring of the secosteroid moiety as well as within the side chain, were designed by Uskokovic (see Zhou et al., 1989). Recently, Posner et al. (1997) developed a new type of vitamin D analogs by modification of the A ring also: Substitution of the 1-hydroxy by a 1-hydroxymethyl group yielded hybrid analogs that combine weak calcemic activity with a still high growth regulatory potential, although they do not bind well to the nuclear vitamin D receptor (VDR) (Peleg et al., 1996).

Colorectal cancer might be particularly susceptible to treatment with vitamin D compounds for the following reasons. Human colon adenocarcinoma Caco-2 cells express the VDR at the mRNA and protein level and, in addition, are able to synthesize its ligand, 1,25-(OH)₂D₃, from its precursor, 25-hydroxyvitamin D₃, due to constitutive expression of a 25-hydroxyvitamin D₃-1-hydroxylase (Cross et al., 1997). In addition, VDR expression in colonic tumor cells is gradually up-regulated in parallel with progression toward malignancy, so that even in poorly differentiated tumors, average VDR density is high (Cross et al., 1996), although individual tumor cells express the receptor to a considerably varying extent (Tong et al., 1998). The efficiency of the 1,25-(OH)₂D₃/VDR system in antagonizing tumor cell growth could thus be specifically enhanced by those vitamin D compounds that could increase the availability of the VDR for endogenously synthesized or systemic 1,25-(OH)₂D₃. In the present study we compared selected vitamin D analogs not only for their antiproliferative and prodifferentiating activity in human colon adenocarcinoma-derived Caco-2 cells, but also for their possible positive effects on VDR expression levels. Because the calcemic activity, particularly of the 1-hydroxymethyl compounds, had been assessed only in an intestinal system (Posner et al., 1992), their potential to mobilize calcium from bone was evaluated from their effects on osteoclast-like cell formation in primary bone marrow cultures.

	Materials and Methods

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Vitamin D Compounds. Synthetic 1,25-(OH)₂D₃ was a gift from Hoffmann-LaRoche (Basel, Switzerland). 1,25-Dihydroxy-16-ene-23-yne-vitamin D₃ (Ro 23-7553) and 1,25-dihydroxy-16-ene-24-oxo-vitamin D₃ (JK-1624-3) were synthesized at Hoffmann-LaRoche (Nutley, NJ). The analogs 1-(hydroxymethyl)-3,25-dihydroxy-16-ene-24-oxovitamin D₃ (JK-1624-2), 1-(hydroxymethyl)-3,25-dihydroxy-16-ene-26,27dihomo vitamin D₃ (JK-1626-2), and 1-(hydroxymethyl)-3,25-dihydroxy-22,24-diene-26,27-dihomo vitamin D₃ (MCW-EE) were synthesized at the Department of Chemistry, The Johns Hopkins University (Baltimore, MD).

Caco-2 Cell Culture. The human colon adenocarcinoma-derived cell line Caco-2 grows in a "tight" monolayer after confluency but displays remarkable heterogeneity in growth and differentiated characteristics (Beaulieu and Quaroni, 1991). Two Caco-2 cell clones, which were analyzed for their proliferative potential and degree of differentiation, were used in the present study. The clone Caco-2/15 was obtained from Dr. A. Quaroni (Cornell University, Ithaca, NY). From this cell line we isolated the subclone Caco-2/AQ by dilution plating after passage 100. The population doubling time of Caco-2/AQ during the logarithmic growth phase was estimated as 24 h versus 36 h of the Caco-2/15 clone (Beaulieu and Quaroni, 1991). The activity of the differentiation marker alkaline phosphatase increased during 20 days of confluent growth from an average of 20 to 60 mU/mg cellular protein in Caco-2/AQ, whereas the respective values for the parent clone Caco-2/15 were 25 and 190 mU/mg protein.

Cell Proliferation Assay. Cells were cultured in DMEM until confluence. Medium was replaced and fresh additions were made every other day. DNA synthesis was assessed by measuring incorporation of [³H]thymidine into cellular DNA. For this purpose cells were incubated for 4 h at 37°C in DMEM containing 4 µCi/ml of [³H]thymidine (specific activity, 70 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO). Cells were then washed with PBS and subsequently fixed and extracted twice with 5% trichloroacetic acid. After two washes with distilled water, cellular protein was solubilized in 1 ml of 0.1 N NaOH. Extracts were assayed for protein (BCA Protein Assay Kit; Pierce, Rockford, IL) and counted for radioactivity.

Alkaline Phosphatase Assay. Cells were rinsed with ice-cold PBS at indicated time points and solubilized with 0.1% Triton X-100 in PBS. The activity was determined with p-nitrophenyl phosphate as substrate on a Microplate reader (MR 7000; Dynatech, West Sussex, England). Enzyme activities were calculated as milliunit per milligram of cellular protein.

Bone Marrow Cell Culture. Eight- to 12-week-old mice (strain HIM:OF1 Swiss, SPF; Institute for Experimental Animal Research of the University of Vienna, Himberg, Austria) were sacrificed by cervical dislocation. Bone marrow cells were prepared from tibiae and femura and cultured as described by Rubin et al. (1992). Briefly, bones were aseptically removed and dissected free of adherent tissue, bone ends were cut off, and the marrow cavity curetted with a sterile 26-gauge needle and flushed with 5 ml of DMEM (supplemented with 1% fetal calf serum, 2.0 mM glutamine, and 1% penicillin/streptomycin). Marrow cells were washed and then suspended in -modified Eagle's medium (Glutamax I, without phenol red, containing 10% fetal calf serum, 1 mM HEPES, and 1% penicillin/streptomycin; Life Technologies, Inc., Grand Island, NY) at 4 × 10⁶ cells/ml. Aliquots (0.5 ml) were plated in 24-well dishes. Hormones were added on day 1 of culture. Two hundred and fifty microliters of medium were replaced by fresh additions every other day. Cultures were performed for 8 days.

Histochemical Determination of Tartrate-Resistant Acid Phosphatase (TRAP). Multinucleated cells were checked for the presence of the osteoclast marker enzyme TRAP. For this purpose, cells were fixed in formaline/acetone/citric acid and reacted for enzyme activity using a commercially available kit (Sigma, Deisenhofen, Germany). Positive cells appeared as dark red. In each of at least three separate experiments, TRAP⁺ multinucleated cells were counted in eight wells per treatment group.

Western Blot Analysis. Cells were rinsed twice with PBS and lysed with boiling lysis buffer (1% SDS, 10 mM Tris, pH 7.4). The lysate was transferred to a microcentrifuge tube and boiled for additional 5 min. Viscosity of samples was reduced by several passages through a 26-gauge needle or by sonication. After centrifugation at 1000 rpm for 5 min at room temperature, the supernatants were stored at 70°C. Protein concentrations were determined using a BCA Protein kit (Pierce).

Data Presentation and Statistical Analysis. Data are presented as means ± S.E. Distribution of results was symmetrical and Student's paired t test was used for statistical evaluation. Significance of difference was assumed when p  .05.

	Results

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Antiproliferative and Prodifferentiating Effects of Vitamin D Compounds in Human Colon Cancer Cells. As in previous studies (Cross et al., 1992, 1993; Bischof et al., 1995), the human colon adenocarcinoma-derived cell line Caco-2 was used to test the antimitotic potency of the newly synthesized 1-hydroxy- and 1-hydroxymethyl vitamin D analogs in comparison with the well known growth inhibitory effects of the natural hormone 1,25-(OH)₂D₃. The chemical structures of all vitamin D compounds tested in the present study are shown in Fig. 1.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Molecular structures of vitamin D compounds used.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Antiproliferative effect of vitamin D compounds on human colon cancer cells. Confluent Caco-2 cells were treated for 4 days with vitamin D sterols at 108 M. In each experiment, [3H]thymidine incorporation was determined in quadruplicates. Data are means ± S.E. from three separate experiments and are expressed as percentage of vehicle control. *, significance of difference from vitamin D-free control at least at p  .05.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Differentiation promoting effect of vitamin D compounds. Confluent Caco-2 cells were treated for 4 days with vitamin D sterols at 108 M. In each experiment, alkaline phosphatase activity was determined in quadruplicates. Data are means ± S.E. from three separate experiments and are expressed as percentage of vehicle control. *, significance of difference from vitamin D-free control at least at p  .01. #, significant difference from vitamin D-free control at least at p  .05.

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View larger version (15K): [in this window] [in a new window]   Fig. 4.   Dose-response relationships for induction of osteoclast-like cells in mouse bone marrow cultures by vitamin D compounds. A, 1-hydroxy compounds; B, 1-hydroxymethyl-compounds.

VDR Protein Expression. The antiproliferative activity of both 1-hydroxy- and 1-hydroxymethyl vitamin D₃ derivatives depends, although for different reasons, on the presence of the VDR. In the case of 1-hydroxy compounds, binding to the VDR, which is mediated by the 1-hydroxy group, seems to be a prerequisite for subsequent VDR-mediated transactivation of gene expression, because 1-deoxy-25-hydroxy vitamin D compounds are devoid of any antimitogenic activity (cf. among others, Bischof et al., 1995). 1-Hydroxymethyl compounds, although showing only a negligible affinity to the VDR if any at all (Peleg et al., 1996), nevertheless require the presence of the VDR for their transactivation activity (Peleg et al., 1998). For these reasons, VDR density could be a critical factor for their antimitogenic efficacy, therefore we evaluated the ability of both types of vitamin D compounds under investigation for homologous regulation of VDR density by Western blot analysis of confluent Caco-2/15 cell lysates after 24 and 48 h treatment at a steroid concentration of 10⁸ M. Figure 5A shows that 1,25-(OH)₂D₃, and also 1,25-(OH)₂-16-ene-23-yne-vitamin D₃ (Ro 25-7553) caused significant up-regulation of the VDR after 24 h of exposure as observed previously (Tong et al., 1998), whereas JK-1624-3 was ineffective. After 48 h however, VDR levels tended to return to control values. Of the 1-hydroxymethyl compounds, JK-1626-2 increased VDR density. The positive effect of JK-1624-2 did not reach statistical significance. In contrast, MCW-EE even significantly reduced VDR levels in Caco-2/15 cells.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   A, Western blot analysis of VDR protein expression in vitamin D-treated Caco-2 cells. B, Western blot analysis of cyclin D1 protein expression in vitamin D-treated Caco-2 cells. Sterols were present in the culture medium for 24, 48, and 96 h at 108 M. Results of densitometric analysis of at least three electropherograms (means ± S.E.). *, significance of difference from vitamin D-free control at least at p  .05.

Cyclin D1. The mechanism by which vitamin D compounds exert their antiproliferative action in colon adenocarcinoma-derived cells is far from well understood. Hulla et al. (1995) presented evidence that in Caco-2 cells, unlike in a variety of other tumor cells, c-myc oncogene expression is resistant to down-regulation by 1,25-(OH)₂D₃. lt is therefore conceivable that inhibition of human colon cancer cell growth by vitamin D requires interaction with a regulatory site downstream of c-myc expression, for which the cell cycle controlling gene, cyclin D1, has been identified as a likely candidate (Tong et al., 1999). Western blot analysis in just confluent Caco-2/15 cell lysates indicates that steady-state levels of cyclin D1 protein expression were reduced by the antiproliferative vitamin D compounds under investigation, with three of six showing highly significant differences to control values (Fig. 5B). lt is interesting, however, that the time dependence of this down-regulation is different for the 1 and 1 compounds: the two most effective prodifferentiating 1 compounds in Caco-2 (i.e., 1,25-(OH)₂D₃ and JK-1624-3) actually require 96 h for maximum reduction of cyclin D1 expression. The two least effective analogs in differentiation of the 1 family apparently act very fast on cyclin D1; after 24 h of exposure expression is already down-regulated by 60%.

	Discussion

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In two previous studies we tested a number of synthetic analogs of 1,25-(OH)₂D₃ for their potency to suppress proliferation of human colon cancer cells. Using the colon adenocarcinoma-derived cell line Caco-2, we were able to confirm that two distinct structural modifications of the secosteroid molecule largely increase the antitumor activity of vitamin D compounds. Of the vitamin D compounds that proved to be equipotent with or even superior to the natural sterol, all bore a double bond at C16 in the D ring in combination with a side chain modification at C23. Thus, compounds like 1,25-(OH)₂-16, 23E-diene-D₃ (Ro 24-2201) or Ro 23-7553, and particularly the C26,27-hexafluoro derivative of the latter (Ro 24-5531), displayed profound growth inhibitory effects and, importantly, at the same time prodifferentiating effects in Caco-2 cell cultures that rendered them potentially useful for colorectal cancer therapy (Cross et al., 1993, Bischof et al., 1995). One result of the present study, side chain oxidation at C24, turned out to be another possibility to improve the antimitotic efficiency of 1,25-(OH)₂D₃ analogs, provided that they display a C16-ene structure. For instance, although 1,25-(OH)₂-16-ene-D₃ (Ro 24-2637) elicited no significant effect on Caco-2 cell replication (Bischof et al., 1995), its C24-oxo derivative, JK-1624-3, reduced [³H]thymidine labeling of DNA, although somewhat less than 1,25-(OH)₂D₃ (Fig. 2).

Comparison of the antiproliferative effects of JK-1624-3 and its 1-(hydroxymethyl) analog, JK-1624-2 (Fig. 2) clearly indicates that by this type of substitution at C1, a substantial growth inhibitory potential is nevertheless retained, although it largely abolishes the binding affinity to the VDR. The observation that the other 1-hydroxymethyl vitamin D compounds tested in the present study (the C26,27-dihomo derivatives JK-1626-2 and MCW-EE) are antimitotically active (Fig. 2), strongly suggests that the intrinsic antiproliferative potential of vitamin D compounds is largely determined by the type of structural modifications induced in the side chain in proper combination with the C16-ene configuration of the D ring in the sterol moiety of the molecule. This may also determine kinetics of the action of vitamin D compounds on cyclin D1 regulation. Therefore the respective transcriptional activity is apparently specifically related to the presence of at least two structural modifications at C16 in the D ring of the steroid moiety and at one or two positions between C22-C26,27 in the side chain.

The mechanism by which vitamin D compounds affect growth of human colon cancer cells has been an enigma (for discussion, see Hulla et al., 1995). Recently, Tong et al. (1999) was able to trace the antimitotic action of 1,25-(OH)₂D₃, in primary cultures of human colorectal cancer cells to the ability of the sterol to suppress mRNA and protein expression levels of cyclin D1. Figure 5B shows that this is valid not only for the 1-(OH) compounds, but also for all 1-hydroxymethyl analogs investigated. The exactly opposite time dependence of MCW-EE and JK-1624-2 compared with 1 compounds is, however, intriguing, especially in view of their low differentiating activity in Caco-2 and bone marrow cells. Although lacking any considerable binding affinity to the VDR, all  compounds, however, nevertheless possess sufficient transcriptional activity toward cyclin D1 gene expression to effectively reduce tumor cell replication.

However, it should be recognized that high-affinity binding to the VDR, although apparently not an absolute requirement for genomic action on colon cancer cell growth, could increase the therapeutic potential of synthetic vitamin D compounds under conditions in which target cell responsiveness is determined by VDR levels. We have demonstrated previously, that during human colon cancer progression, VDR expression is elevated in the early stages, only to drop to low levels during the late stages (Cross et al., 1996).

Figure 2 also illustrates the fact that 1-hydroxymethyl-vitamin D analogs exert their antiproliferative activity largely independent from VDR expression levels in the target cells. There is considerable antiproliferative activity of 1-hydroxymethyl-vitamin D analogs, although MCW-EE actually reduces VDR expression (cf. Fig. 5A).

In conclusion, our data clearly indicate that because of certain structural modifications of the C ring and the side chain, 1-hydroxymethyl-vitamin D "hybrid" analogs show significant antiproliferative activity in human colon cancer cells, which, for example in the case of JK-1626-2, is comparable with that of some of the widely tested 1-(OH) analogs such as Ro 23-7553 (Fig. 2). Surprisingly, JK-1626-2 also has prodifferentiating activity, whereas the other hybrid analogs tested (JK-1624-2 and MCW-EE) are rather ineffective in this respect (cf. Figs. 3 and 4). This, however, would be consistent with the assumption that substitution of the 1-hydroxy- by a 1-hydroxymethyl group results in dissociation between antiproliferative activity and effects on differentiated cell functions, e.g., induction of calbindin_28k-mediated intestinal calcium transport (Posner et al., 1992). At the moment we have no explanation why the 1-hydroxymethyl analog JK-1626-2 nevertheless induces phenotypic differentiation of Caco-2 cells and osteoclast-like cell formation, except that the particular configuration of its side chain at C26 and 27 (cf. Fig. 1) overcomes some effects of the 1-hydroxymethyl group. Also, the fact that side chain modifications can confer higher prodifferentiating activity to vitamin D compounds can be deduced from the observation that the 24-oxo metabolites of a number of 1-hydroxy vitamin D analogs have a higher capacity to induce differentiation in breast cancer cells than their parent compounds, although both groups are similarly active in inhibiting clonal proliferation (Campbell et al., 1997). In any case, the lack of prodifferentiating activity in the two hybrid analogs tested could even be seen as advantageous for their possible use in cancer therapy, because both compounds are largely ineffective in promoting osteoclast differentiation (cf. Fig. 4) and could thus be administered even at dose levels that otherwise would cause hypercalcemia from enhanced osteoclastic bone resorption.