E B 1089, A N O V E L V I T A M I N D A N A L O G U E , H A S
S T R O N G A N T I P R O L I F E R A T I V E A N D D I F F E R E N T I A T I O N
I N D U C I N G E F F E C T S O N C A N C E R C E L L S
IDA S. MATHIASEN, 1. KAY W. COLSTON 2 and LxSE BINDERUP 1 1Department of Biology, LEO Pharmaceutical Products, 2750 Ballerup, Denmark and 2Department of Clinical Biochemistry, St Georges Hospital Medical School,
London SW17 ORE, England
(Received 3 November 1992; accepted 26 May 1993)
Summary--EB 1089 is a novel vitamin D analogue which in vitro strongly inhibits the proliferation of U937 histiocytic lymphoma cells and MCF-7 breast cancer cells, with a potency of 50 to 100 times that of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Studies of c-myc and c-fos expression in MCF-7 cells and of differentiation markers in U937 cells show that growth inhibition by EB 1089 is accompanied by induction of differentiation. The ability of EB 1089 to affect calcium metabolism in vivo in rats is decreased, compared to 1,25(OH)ED 3. This low calcemic effect combined with the strong biological effect on cancer cells in vitro, makes EB 1089 an interesting candidate for treatment of cancer.
INTRODUCTION
The physiologically active metabolite of vitamin D3 [1,25-dihydroxyvitamin D3; 1,25(OH)2D3] regulates calcium and phosphate metabolism, primarily by effects on intestine, bone and kidney [1, 2]. In recent years, 1,25(OH):D3 has been shown to possess cell-regulating properties in a number of tissues and cells, not directly involved in intestinal calcium absorption and bone mineralization [3]. Notably, 1,25(OH)2D 3 is able to inhibit cell proliferation and induce differentiation in a number of cancer cell lines [4~6].
The effects of 1,25(OH)2D3 on cell growth and differentiation are believed to be mediated via binding to a specific receptor [7]. This recep- tor is located intracellularly and belongs to the family of classical steroid receptors [8]. The vita- min D receptor (VDR) binds 1,25(OH)2D3 with high affinity, interacts with specific sequences of DNA and alters gene expression [9].
Consistent with these observations, it has been shown that 1,25(OH)2D3 downregulates c-myc and induces a burst of c-fos expression in a number of cultured cells [10-12]. In vivo, administration of 1,25(OH)2D3 or its synthetic prodrug l-~-hydroxyvitamin D3 to tumour- bearing animals prolongs their survival,
*To whom correspondence should be addressed.
induces regression of already existing tumours and/or inhibits metastatic spread of tumour cells[13-15]. These observations suggest that 1,25(OH)2D3 may be of interest in the therapy of cancer. However, the use of 1,25(OH)2D3 is severely limited by its potent effects on calcium metabolism, with the risk of inducing side effects such as hypercalcemia and soft tissue calcifica- tions. New vitamin D analogues with increased ability to regulate cancer cell growth but with a reduced risk of causing calcemic side effects are therefore of great interest. Three such analogues have recently been described: 16-ene-23-yne- 1,25(OH)2D3, which prolongs the survival of leukemic mice [16]; 22-oxa-l,25(OH)2D3, which inhibits cancer cell proliferation in vitro [17] and EB 1089 which causes regression of carcinogen- induced mammary tumours in rats[18]. All three compounds have been shown to inhibit cancer cell proliferation in vitro, the first two with 2 to 10 times the potency of 1,25(OH)2D 3, whereas EB 1089 has been shown to be approx. 10 to 60 times more potent [19, 20].
In the present study EB 1089 was investigated for its effects on cell proliferation and differen- tiation in two human cancer cell lines: the histiocytic lymphoma cell line U937 and the human mammary carcinoma cell line MCF-7. Both cell lines display high affinity receptors for 1,25(OH)2D3 [21,22]. Induction of cell differentiation was studied in the U937 cells, by
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366 IDA S. MATHIASEN et al.
OH
HO~]~OH 1 ,25(OH)2D 3 EB 1 0 8 9
Fig. 1. The chemical identity of 1,25(OH)2D 3 and EB 1089.
assessing their maturation along the monocyte- macrophage pathway. In MCF-7 cells, c-myc and c-fos expression was measured by Northern blot analysis. In addition, binding of EB 1089 to VDR from MCF-7 cells was studied. The effects of EB 1089 on calcium metabolism in vivo were investigated after p.o. and i.p. adminis- tration to rats. In all studies, 1,25(OH)2D3 was used as a reference compound. The struc- tures of 1,25(OH)2D3 and EB 1089 are given in Fig. 1.
MATERIALS AND METHODS
Cell cultures
MCF-7 (Tumorbank DKZF; Heidelberg, Germany) and U937 (ATCC; Rocheville, U.S.A.) cells were propagated in culture flasks and on multiwell plates (Nunc, Roskilde, Den- mark). Growth medium for routine propagation was for MCF-7: DMEM w/o phenolred (GIBCO, Grand Island, U.S.A.) supplemented with 5% foetal calf serum (FCS) (GIBCO), 2 mM L-glutamine (Sigma, St Louis, U.S.A.), 100IU/ml penicillin-streptomycin (pen/strep, GIBCO) and for U937: RPMI 1640 (GIBCO), 10% FCS, 2mM L-glutamine and pen/strep.
Prior to growth experiments MCF-7 cells were grown in DMEM supplemented with 5% charcoal stripped FCS, 2 mM L-glutamine and pen/strep for 1 week, and passaged to multiwell plates in the same medium, in the absence or presence of test compounds.
Compounds
1,25(OH)2D3 and EB 1089123] were syn- thesized in the Department of Chemical Research (Leo Pharmaceutical Products, Den- mark). For the in vitro studies both were diluted in 2-propanol and added to the medium at a max. concentration of 0.25%. For the in vivo studies compounds were dissolved in propylene glycol for oral administration, and in propylene
glycol, 0.05 M Na2HPO4 (80:20) for intra- peritoneal administration.
In vitro experiments with U937
Cells were seeded at 1 × 105 cells/ml in culture flasks at 5ml cells/flask. 1,25(OH)2D 3 or EB 1089 were added in the concentrations indicated in the Results section. Control cultures received the diluent, 2-propanol. After 96 h, proliferation was assessed by counting of cells, and IC50 was calculated from the dose-response curves. Induction of differentiation was followed by the appearance of membrane-associated nonspecific esterase activity as described previously[24]. Four separate cultures were made for each concentration tested.
In vitro experiments with MCF-7
Cell culture. Cells were seeded at a cell density of 1.5 × 104 cells/ml medium in multiwell plates and 1,25(OH)2D 3 or EB 1089 were added in the concentrations indicated in the Results section. Control cultures received the diluent, 2-propanol. At day 6, thymidine incorpora- tion was measured by labelling for 4h with 37 kBq/ml [3H]thymidine (185 GBq/mmol, Amersham, England) in medium obtained fresh the day before. At the end of the incubation period, medium was removed and cells were washed with icecold 0.9% NaC1 containing 25mg/ml thymidine thereafter with icecold PBS. Cells were solubilized in 0.5 M NaOH and counted in a liquid scintillation counter. Five separate cultures were made for each con- centration tested.
Receptor studies. These were performed in a crude extract prepared as described pre- viously [25] with small modifications: cells rou- tinely propagated were scraped off with a rubber policeman just before confluence keeping the cells icecold. Cells were centrifuged at 2500 g for 5 min at 4°C and resuspended in TKEDM- buffer (10mM Tris-HCl, 0.3M KC1, 1 mM EDTA, 5 mM dithiothreitol, 10 mM Na2MoO4) and adjusted to 5 × 107 cells/ml. Cells were lysed by three times freeze/thaw, centrifuged for 1 h at 105,000g at 4°C and the supernatant was used for receptor studies. The protein concentration was adjusted to 3 mg/ml and binding assay was performed as in [24].
Northern blots. Northern blots of the expression of c-myc and c-fos was performed as follows: total RNA was isolated from MCF-7 cells by guanidine thiocyanate extraction fol- lowed by phenol/chloroform extraction [26] and
Effects o f a novel v i t amin D ana logue , 1 EB 1089
Table I. Growth and differentiation of U937 cells in vitro
Inhibition Esterase activity of Compound of cell Conc. nonadherent cells tested proliferation in assay (M) (% positive cells)
U937 cells (1 x 105 cells/ml) were cultured for 96h in the presence of 1,25(OH)2 D 3 or EB 1089. At the end of the experiment, cell proliferation was determined by cell counting. Nonadherent cells were stained for esterase activity as a marker for differentiation. Values represent the mean + SD of 3 separate experiments.
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was separated on a 1.2% agarose and 0.66 M formaldehyde denaturing gel [27]. Samples were transferred to Hybond N + membrane (Amersham) and hybridized to human c-myc or human c-fos oligonucleotides (Oncogene Science, U.S.A.) which had been 5' end labelled with [32p]ATP (>5000 Ci/mmol; Amersham). Blots were washed and autoradiographed with intensifying screens.
Effects of calcium metabolism in vivo
In vivo effects of EB 1089 on calcium metabolism were studied in inbred female Lewis rats (140-150 g). They were treated with 1,25(OH)2D 3 (0.5/~g/kg/day) or EB 1089 (0.1, 0.5, 1.0 and 2.Spg/kg/day). The compounds were administered p.o. or i.p., for 7 days. Each group consisted of 3 rats. Urine was collected daily and serum was taken at the end of the experiment as described previously[24]. Calcium was determined in urine and serum by complex formation with o-cresolphthalein (Boehringer Mannheim, Germany). The results are expressed as mean values _+ SD. Statist- ical analysis was done using paired samples t-test.
Growth studies were also performed with the mammary carcinoma cell line MCF-7. The MCF-7 cells were grown in steroid-deficient medium (charcoal stripped serum) for 6 days with 1,25(OH)2D 3 or EB 1089, prior to assessment of thymidine incorporation. The in- hibitory effect of EB 1089 on MCF-7 cell pro- liferation was approx. 50 times stronger than that of 1,25(OH)2D 3. The concentration result- ing in 50% inhibition (ICs0) was 1.0 x 10 -s M for 1,25(OH)2D a and 2.3 x 10-1°M for EB 1089.
Receptor binding in MCF-7 cells
The ability of EB 1089 to bind to the intra- cellular VDR in MCF-7 cells was studied next. Figure 2 shows displacement curves of 1,25(OH)ED3 and EB 1089 displacing 3H- labelled 1,25(OH)2D 3 from the receptor. Fifty percent displacement was obtained with 1,25(OH)2D 3 at 5 x 10-13M and with EB 1089 at 2 x 10 -11M. EB 1089 thus has approx. 40
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100
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R E S U L T S ~ eo
Effects of EB 1089 on cell growth and differen- N
tiation in vitro 40
The effects of EB 1089 on cell proliferation 20
and differentiation were first discovered in a routine screening programme of vitamin D 0 analogues in cultures of human lymphoma cells U937. Table 1 shows that EB 1089 was approx. 100 times more potent in exerting antiproliferative effects and in inducing differen- tiation of U937 cells than 1,25(OH)2D 3 itself.
1 ,25 B 1 0 8 9
10 "Is 10 -t4 10 "lz 10 - lz 10 " t l 10 "1° 10 - I 10"
comoet i tor | M I
Fig. 2. D i sp lacemen t of [3H]I,25(OH)2D3 f rom the V D R f rom M C F - 7 wi th increas ing a m o u n t s of un labe l led 1,25(OH)2D3 ( l l ) or EB 1089 (A) . Each curve is based on
the m e a n of five separa te exper iments .
368 IDA S. MATHIASEN et al.
A
0.30 • Scatchard plot of VDR binding 1 , 2 5 ( O H ) 2 D s
Fig. 3. Scatchard plots of receptor binding to VDR of 1,25(OH)2D 3 (A) and EB 1089 (B). The data from the displacement studies in Fig. 2 is here presented as Scatchard plots, where the negative reciprocal slope is equivalent to the
K o value (disassociation constant) for receptor binding.
times lower affinity for the VDR than 1,25(OH)2D3, the naturally occurring ligand. These values are in accordance with the Ko values found in the Scatchard plots shown in Fig. 3(A and B).
This relatively low affinity is in contrast to the observed strong antiproliferative effect of EB 1089. To know if EB 1089 despite the lower binding affinity to the VDR is able to affect expression of genes involved in the regulation of cell proliferation and differentiation, studies of c-myc and c-fos expression in MCF-7 cells were undertaken.
Expression of c-myc and c-los in MCF- 7 cells
The expression of c-myc was followed after different periods of incubation with 1,25(OH)2D3. Figure 4(A) shows that the expression of c-myc mRNA is inhibited by 5 x 10-SM 1,25(OH)2D 3 after 2.5h, in
accordance with previously described obser- vations [12].
A similar experiment was performed to find the time dependent expression of c-fos [Fig. 4(B)]. A burst of c-fos mRNA appeared after 15 min, reached a peak after 1 h and then declined to under detection level, making the timepoint 1 h the most sensitive.
Subsequently, RNA was extracted from cells treated with various concentrations of EB 1089 and 1,25(OH)2D 3. Based on the results above, c-myc expression was measured after 3 h and c-fos expression after 1 h of incubation [Fig. 5(A and B), respectively]. Both genes show a dose dependent regulation of expression, indicating that EB 1089 was approx. 50 times more potent than 1,25(OH)2D3.
Calcium metabolism in vivo
The calcemic effects of EB 1089 and 1,25(OH)2D3 were measured in rats after p.o. and i.p. administration for 7 days. Calcium excretion in urine was assessed daily and serum calcium levels were measured on day 7. Figure 6 shows the urine calcium excretion after p.o. administration of EB 1089 (0.1, 0.5, 1.0 and 2.5 pg/kg/day) in comparison to vehicle- treated rats and rats treated with 1,25(OH)2D3 (0.5 pg/kg/day). EB 1089 was found to have half the potency of 1,25(OH)2Ds. Analysis of serum calcium levels showed a similar picture (results not shown). The lower calcemic activity of EB 1089 after p.o. administration could be caused by a decreased absorption. After i.p. adminis- tration EB 1089 was however also less potent than 1,25(OH)2D 3 (results not shown).
DISCUSSION
The proliferation of two human cancer cell lines, the histiocytic lymphoma U937 and the mammary carcinoma MCF-7, is potently inhibited by the new vitamin D analogue EB 1089, confirming previous studies in these cell lines [20, 19, 28]. The antiproliferative effect was found to be 50 to 100 times stronger than that exerted by 1,25(OH)2D3. In addition, EB 1089 was also found to be a potent inducer of cell differentiation. In U937 cells differentiation occurred along the monocyte-macrophage pathway. In MCF-7 cells, induction of cell differentiation is more difficult to assess. In the present study we have chosen to follow changes in the expression of the c-myc and c-fos proto- oncogenes, previously shown to be regulated
Effects of a novel vitamin D analogue, 1 EB 1089
A C 15rain 30min lh 2.5h 7h
369
c-myc
rRNA 18S
B C 15min 30min lh 2.5h 7h
.... ~ ~ ~ i ~ ¸ • ~ ~ . . . . i~ii~ ~ ' ~ • i i i~ ~iiii~!ii~!~'~!~ii! ~̧~ ~i~i~i i i~ '~i i i i l i~ i~i!i~i~!~ ii
c- los
rRNA 18S
Fig. 4. Northern blots of the time dependent of c-myc (A) and c-fos (B) in MCF-7 cells. 1,25(OH)2D 3 is added at 5 x I0 -s M and RNA is extracted at the indicated times, c-myc expression is sensitive to
1,25(OH)2D 3 2.5 h after addition and c-fos expression between 30 min and 1 h.
by 1,25(OH)2D 3[11, 12]. EB 1089 decreased the expression of c-myc mRNA and transiently increased c-fos expression, being approx. 50 times more potent than 1,25(OH)2D 3.
Thus, changes in cell proliferation and differ- entiation induced by EB 1089 correlated well in the two cell types studied. As these changes are considered to be under genomic control it was rather surprising to find that EB 1089 had a lower affinity than 1,25(OH)2D3 for the VDR in MCF-7 cells. This finding suggests that different types of receptors may exist in the MCF-7 cells. In contrast to 1,25(OH)2D 3, EB 1089 do not show the same disparity between the concentration giving 50% displacement from VDR and that giving 50% inhibition of cell growth. This finding suggests that differ- ences in signal transduction exist between the two compounds. However no evidence for two different receptors was observed in the Scatchard plots from the receptor studies. Alter- natively, the entrance rate into the cells of the two compounds could be different due to the presence of vitamin D binding protein (DBP) in the serum. This protein could bind the two compounds with different affinities and thereby cause differences in the availability of free
compounds, as described for other vitamin D analogues [29].
The in vivo studies of calcium metabolism show that EB 1089 has a slightly lower ability to affect calcium uptake and excretion than 1,25(OH)2 D 3 . This effect was not due to differ- ences in the absorption of the two compounds, as p.o. and i.p. administration gave similar results, but can of course be due to differences in metabolic clearance. However, previous stud- ies have shown that EB 1089 and 1,25(OH)2D3 have similar half-lives in rats [20]. An interesting possibility is that the relatively low calcemic effect of EB 1089 (compared to its high in vitro activity in cancer cells) is related to its affinity for the VDR. It would thus be interesting to assess whether EB 1089 is able to induce 1,25(OH)2D3-regulated genes such as the cal- bindin and osteocalcin genes, important in the regulation of intestinal calcium absorption and bone formation.
In conclusion, EB 1089 is a novel vitamin D analogue which may be of interest in elucidating mechanisms underlying the regulation of cancer cell growth and differentiation. Additionally, EB 1089 may be of potential value in cancer
370
A
C
IDA S. MATHIASEN et al.
1,25(OH)2D a EB 1089
5 x 1 0 -a 5 x 1 0 -g 5 x 1 0 -Ig 5 x 1 0 -~o 5 x 1 0 -~I
c-myc
rRNA 18S
B 1 , 2 5 ( O H ) 2 D a E B 1 0 8 9
C 5 x 1 0 -s 5 x 1 0 -9 5 x 1 0 - I ° 5 x 1 0 - I ° 5 x 1 0 -~+
c-fos
rRNA 18S
Fig. 5. Northern blots of the dose dependent expression of c-myc (A) and c-fos (B). MCF-7 cells are given various concentrations of either 1,25(OH)2D 3 or EB 1089. C: control, 1 : 5 × 10 -s, 2 : 5 x 10 -9, 3:
5 × 10- t°M 1,25(OH)2D3, 4 :5 × 10 -l°, 5 :5 x 10 -It M EB 1089.
II "U
--~ 200 - o E
tu 15o - z n- - I z 1oo-
- 5 0 o . . I < O
o
CONTROL
[-7 o
CALCIUM METABOLISM IN VIVO
1,25(OH)z,,D z EB 1089
0.5 0.1 0.5 1.0 2.5
D O S A G E ( IJg lkg lday p.o.)
Fig. 6. Calcium excretion in urine. Rats were treated with vehicle (open bar), 1,25(OH)2D 3 (hatched bar) or EB 1089 (crossed-hatched bars) p.o., daily for 7 days. All treated groups were compared to the groups of control rats (paired t-test). Asterisks indicate P < 0.05. Values represent the
means of 2-4 separate experiments.
therapy in view of its relatively low calcemic activity, its ability to regulate tumour cell growth in vitro and its previously described antitumour effects in rats with mammary carci- nomas [18, 28].
Acknowledgement--The authors thank Mrs Bente Hasselriis for her expert technical assistance.
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