antiproliferative response of both cell lines to 1,25(OH)2D3.9-cis-RA exerted antiproliferative ... ment of the human colon cancer cell lines HT-29 and Caco-2 with.
ICANCER RESEARCH56. 623-632, Febniary I. 19961
Antiproliferative Responses of Two Human Colon Cancer Cell Lines to Vitamin D3 Are Differentially Modified by 9-cis-Retinoic Acid1 Katherine F. Kane,2Michael j S. Langman, and Graham R. Williams3 Department of Medicine, The Queen Elizabeth Hospital, University of Birmingham. Edgbaston, Birmingham B15 2TH, United Kingdom
ABSTRACT la,25-Dihydroxyvitamin D3 [1,25(OH)2D3]exerts antiproliferative ac tions in colorectal cancer, but their underlying molecular mechanisms have not been determined. 1,25(OH)2D3regulates target gene transcrip lion via a specific nuclear Vitamin D receptor (VDR), which mediates hormone action preferentially as a heterodimer with 9-cis-retlnoic acid receptors
(RXRs).
retinoic
acid
(RA)
We investigated in two
the actions
human
colon
of 1,25(OH)2D3
cancer
cell
lines,
and 9-d.c. HT-29
and
Caco-2. Both expressed mRNAs encoding VDR, RXRa, and RXR'y, and
VDR wasregulated posttranscriptionally inCaco-2 cellsThere wasan antiproliferative response of both cell lines to 1,25(OH)2D3. 9-cis-RA exerted antiproliferative effects on Caco-2 cells but blocked 1,25(OH)2D3 actions in HT-29 cells. The 1,25(OH)2D3-responsivegene 25-hydroxyvita mm D3 24-hydroxylase was induced in both cell lines by 1,25(OH)2D3but in only HT-29 cells by 9-cis-RA. 1,25(OH)2D3and 9-cis-RA cotreatment enhanced 24-hydroxylase expression in HT-29 cells only. The 24-hydrox ylase enzyme is known to result in catabolism of 1,25(OH)2D3and atten uation of its actions. Increased 24-hydroxylase activity in HT-29 cells, but not in Caco-2 cells, in response to 9-cis-RA may account for some of the complex cell-specific responses demonstrated in these studies.
INTRODUCTION The actions of l,25(OH)2D34 in bone, kidney, and small intestine, which control Ca2@ and P043 homeostasis, are well established. More recently, important antiproliferative effects of l,25(OH)2D3 have been demonstrated by the induction of differentiation in many normal and malignant target cells (1—5).These findings suggest a role for l,25(OH)2D3 in the prevention and treatment of a variety of tumors. Epidemiological studies indicate that vitamin D3 confers significant protection against coborectal cancer when assessed by dietary intake (6) or serum levels (7). Similar effects for vitamin D3 have been demonstrated in experimental models of colon cancer (8—12).Treat ment of the human colon cancer cell lines HT-29 and Caco-2 with 1,25(OH)2D3 induces differentiation and inhibits cell proliferation in vitro (13—17).Vitamin D3 analogues, with low calcemic activity relative to their differentiation potency, have been synthesized. These agents possess promising antiprobiferative properties in vitro (1, 3, 18, 19) and are the focus of several clinical trials. The actions of l,25(OH)2D3 are mediated by its high-affinity nu clear receptor (VDR), which is a member of the steroid and thyroid hormone receptor superfamily (20, 21). VDR binds to specific target sequences of DNA, l,25(OH)2D3 response elements, and functions as
@
whom
requests
for
reprints
should
be addressed.
Phone:
44
121 472
131 1; Fax:
Address:
Department
of Molecular
Medicine,
Royal
Postgraduate
abbreviations
used
are:
l,25(OH)2D3,
la,25-dihydroxyvitamin
D3;
VDR
has been
detected
cobonocyte
cultures
derived
the target gene 24-hydroxylase
throughout
the normal
human
from human
adenocarcinomas;
is induced in these colonocytes by
l,25(OH)2D3 (3 1). 24-Hydroxylase is a cytochrome P450 enzyme, which hydroxylates l,25(OH)2D3 at the 24 position, resulting in a metabobite of markedly reduced activity. Thus, l,25(OH)2D3 induc tion of 24-hydroxylase causes attenuation of its own actions (32). 9-cis-RA exerts antiproliferative actions in in vitro models of breast cancer and has been proposed as a chemotherapeutic agent (33). There are no epidemiological data that link vitamin A or retinoid status with colorectal carcinogenesis. However, f3-carotene, a vitamin A precur sor, inhibits mucosal omithine decarboxylase activity in the rectum of colon cancer patients (34), and retinoids protect against azoxymeth ane-induced tumors in rats (35). The actions of 9-cis-RA have not been studied in the colon. The aim of these studies was to determine whether 9-cis-RA modified l,25(OH)2D3 action in two human colon cancer cell lines (HT-29 and Caco-2) that express VDR. We characterized the mdc pendent effects of l,25(OH)2D3 and 9-cis-RA on cell growth, VDR and RXR expression, and 24-hydroxylase gene regulation. Finally, the modulatory effects of 9-cis-RA on each of the l,25(OH)2D3 responses were determined in both cell lines. MATERIALS
AND METHODS
HT—29 and Caco-2 Cell Culture and Reagents. HT-29 andCaco-2 cells (European
catalogue
of animal
cell
cultures)
were
maintained
in DMEM
supplemented with glutamax-l(L-alanyl-L-glutamine) and 10% (HT-29) or 20% (Caco-2)
FCS (GIBCO,
supplemented
l,25(OH)2D3,
44
Paisley,
United
Kingdom).
The concentration
of
9-cis-RA,
with
10% CSS
or combinations
for 24 h prior of both.
to treatment
l,25(OH)2D3
with
was prepared
from 4 mM stock solutions in isopropanol, and 9-cis-RA was prepared from powder dissolved in ethanol (1 mM). Cell viability, assessed by trypan blue exclusion, was greater than 90% in all experiments. Growth Assessment. HT-29 and Caco-2 cells were seeded (1 X l0@
Medical
VDR,
primary
in DMEM
School, Hammersmith Hospital, Du Cane Road, London Wl2 ONN, United Kingdom 4 The
(27).
CSS(36),in whichl,25(OH)2D3levelswereundetectable.Cellswerecultured
121627 2384. 3 Present
tab mucosa
colon using immunocytochemical (28) and radioligand binding tech niques (29). We have demonstrated expression of mRNAs encoding VDR, RXRa, and RXR7 in both normal and malignant human cob rectal tissue (30), and it has been suggested that colonic tumor expression of VDR mRNA may predict clinical outcome (17). In addition, we have shown that VDR is expressed and functional in
l,25(OH)2D3 in FCS was 100 pmol/liter (l0 ‘° M) when assessed by RIA following HPLC. Cell treatments were performed in medium supplemented with Dowex l-X2—400(Sigma Chemical Co., Poole, United Kingdom) and
Received 8/2/95; accepted I 1/22/95. 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. Supported by grants from the West Midlands Regional Health Authority and the Medical Research Council (United Kingdom). 2 To
a ligand-inducible transcription factor to regulate target gene expres sion (21). VDR, together with the receptors for thyroid hormone (T3Rs) and abl-trans-RA (RARs), form a subgroup of promiscuous receptors, which bind to DNA as heterodimers with receptors for 9-cis-RA (RXR; Refs. 22—24).There are three RXR genes (RXRa, RXRf3, and RXRy), which encode multiple-receptor isoforms to pro vide the potential for complex modification of l,25(OH)2D3 signaling by 9-cis-RA (23—26). l,25(OH)2D3 exerts antiproliferative actions in normal human rec
vita
mm D receptor; T3R, triiodothyronine receptor; RA, retinoic acid; RXR, retinoid X receptor; 25(OH)D3, 25-hydroxyvitamin D3; 24-hydroxylase, 2S(OH)D3 24-hydroxylase; HPLC, high-performance liquid chromatography; CSS, charcoal-stripped FCS; PMSF, phenylmethylsulfonyl fluoride.
cells/ml)
in 24-well
plates and treated
for 72 h with l,25(OH)2D3
or 9-cis-RA
(1 X l0 ‘@—l X l0-@ M). Vehicle in the medium did not exceed 0.01%, a concentration 623
that did not affect
cell growth
or viability
(data
not shown).
ANTIPROLIFERATIVEACTIONS OF VITAMIN D3 AND 9-cis-RA
HT-29 cells were harvested for assessment of proliferation by Coulter counting (Coulter Electronics, Ltd., Bedfordshire, United Kingdom) and/or [3H]thymi
PBS containing unlabeled thymidine (100 pM), and cells were incubated for a
192 mxi glycine, and 20% (v/v) methanol]. Membranes were blocked (1 h, 25°C)in PBS-T (PBS plus 0. 1% Tween-20; Sigma) containing 20% (w/v) nonfat milk powder (Marvel; Premier Brands, Stafford, United Kingdom) and rinsed twice in PBS, followed by a further wash in PBS-T for 15 mm. Filters were incubated overnight with a primary rat monoclonal antibody raised against chicken VDR protein (Cambridge Research Biochemicals, Zeneca,
further
Northwich,
dine
(Amersham
International,
Buckingham,
United
Kingdom)
uptake.
[3H]thymidine uptake was determined after incubation of cells with [3H]thy midine
(0.1
@zCiJml, 37°C, 1 h). Subsequently,
5 mm, washed
roacetic
acid
twice
the medium
in PBS, and precipitated
(4°C, 30 mm).
Trichloroacetic
was replaced
with
acid
was
with
I ml 5% trichlo
removed,
and
cell
precipitates were solubilized in 0. 1 M NaOH prior to counting in 4 ml Ecoscint
A aqueous scintillation fluid (National Diagnostics, Atlanta, GA). RNA Extraction and Northern Analysis of Receptor and 24-Hydroxy lase mRNAs. HT-29 and Caco-2 cells were treated with l,25(OH)2D3,9-cis RA (l0@ or iO@ M),or vehicle for 6 or 24 h prior to harvest. Total cellular RNA was extracted (37), and the mRNA fraction was enriched by oligode oxythymidine column chromatography [oligo (dl) cellulose type 7; Pharmacia LKB Biotechnology AB, St. Albans, United Kingdom; Refs. 3 and 38]. Polyadenylated mRNA (—3 @g)or total RNA (15 @zg)was loaded into a denaturing 1.2% formaldehyde agarose gel and resolved by electrophoresis (100 V. 4 h; Ref. 38). RNA was transferred overnight to Hybond N@mem branes (Amersham) and bound covalently to filters by UV irradiation (245 nm, 120 millijoules). Filters were prehybndized (8 h) and hybridized (16 h) at 65°C with 32P-labeled cDNA or cRNA probes in phosphate [0.77 M NaH2PO4/
Na2HPO4(pH 7.2), 5 mMEDTA, 7% SDS, and 100 @ig/mlsonicated salmon sperm DNA] or formamide
[50% deionized
formamide,
5 X saline-sodium
United
Kingdom;
Ref.
43) diluted
1:1000
in PBS-T
(0.05%).
Following three 10-mm washes in PBS-T, filters were incubated with the secondary antibody (horseradish peroxidase-conjugated antirat; Amersham) diluted 1:50,000 in PBS-T (0.05%) for 90 mm at 25°C,and three further washes were performed (10 mm each in PBS-T). Specific receptor proteins were detected by enhanced chemiluminescence (Amersham) after exposure of the filters to X-ray film for 15 mm, and quantitation by laser densitometry was performed.
Control
experiments
included
omission
of the primary
or secondary
antibody and the use of enhanced chemiluminescence detection alone. No protein bands were detected in these controls in either cell line. In addition, quantitation
experiments
were performed,
which
demonstrated
that there was
a linear relationship between densitometry readings and the amount of VDR detected
across
a range of nuclear
protein
concentrations.
Assay of 24-Hydroxylase Activity. Cells were seeded in six-well plates (1 X l0@cells/mi), pretreated for 24 h in DMEM and CSS, and then incubated in 2 ml serum-free
DMEM
supplemented
with 1.5 mg/mI BSA and hormones.
Cells were treated for 24 h with l,25(OH)2D3(l0 ‘ ‘—l0@ M)or vehicle only in the presence
or absence
of 9-cis-RA
(l0@
M). Following
treatment,
25,000
phosphate-EDTA (20X SSPE = NaCl, 175.3g; NaH2PO4H2O,27.6g; EDTA,
dpm/well
7.4g. pH 7.4, in 1 liter), 0.15 M Tris (pH 8), 1% SDS, 5X Denhardt's
25(OH)D3were added to each well and incubated for a further 3 h. Media were removed for analysis, and the cells were trypsinized for counting. l,25(OH)2D3 metabolites were extracted from the medium in 5 ml chloroform:methanol [3:2 (v:v)] by vortexing for 15 s. The aqueous and organic layers were separated by centrifugation (10 mm, 4°C,2500 rpm). The organic phase was collected, dried under N2, resuspended in 100% ethanol, and stored at —20°C under N2 prior to analysis by normal-phase HPLC (44).
solution
(50X Denhart's = 5 g ftcoli 400, 5 g polyvinylpyrrolidone, 5 g BSA in 500 ml), and 100 @zg/mlsonicated salmon sperm DNA] buffers. Filters were washed at high stringency [0.1X SSC (20X SSC 3 M NaCI, 0.3 M Na3 citrate2 H,O, pH 7.0) and 0. 1% SDS at 65°Cfor 1 h (cDNA probes) or 75°C
for 1 h (cRNA probes)] prior to autoradiography for 6 h—28 days, depending on the probe used. Autoradiographs were quantified by laser densitometry (LKB 2202 Ultroscan laser densitometer; LKB, Bromma, Sweden; Hewlett Packard-LKB 3390A reporting integrator; Hewlett Packard, Avondale, PA), and receptor mRNA expression was standardized relative to @3-actin. Probes.
A
full-length
mouse
@3-actin cDNA
was
radiolabeled
with
[a-32P]dCTP (Amersham) by nick translation (Amersham; Ref. 38). cRNA probes
were
(Riboprobe
labeled system;
with
[32P]UTP
Promega,
(Amersham)
Southampton,
United
by in vitro Kingdom)
transcription
using T7 or T3
bacteriophage RNA polymerase. cRNAs were synthesized from a full-length human VDR cDNA (39) subcloned into pBluescript KS@(Stratagene, Cam bridge,
United
Kingdom)
and linearized
with HindIII
and from
full-length
human RXRa (40) and mouse RXRf3 and RXR'y cDNAs (41) cloned into pBluescript SK@(Stratagene) and linearized with XbaI, XbaI, or XhoI, respec tively. A human 24-hydroxylase cDNA probe (42) was labeled with [a-32P]dCTP by the random primer method (Pharmacia LKB, St. Albans, Nuclear Protein Preparation. HT-29 and Caco-2 cells were subcultured flasks
to 70% confluence
and transferred
to CSS-supplemented
media for 24 h prior to treatment with l,25(OH)2D3 or 9-cis-RA (l0 M) for a a further
24 h. Cells
were
trypsinized,
washed
with
PBS,
and
(800 X g, 4°C,10 mm), resuspended in 3.5 ml STM-PMSF containing 0.5%
Triton X-lOO (Sigma), and incubated on ice for 10 mm. This process was repeated twice, and the resultant nuclear pellet was resuspended in 300 pA STM-PMSF containing 5 msi DTT, 0.4 M KC1,and 20% (v/v) glycerol. The was agitated
prior to centrifugation
on ice for 15 mm to extract (2900
soluble
nuclear
stacking
HPLC
was performed
25
using
ng/ml
a Zorbax-Sil
unlabeled
column
derivatives
were chromatographed,
and retention
times
were
determined
by
Uv absorption at 265 nm or by scintillation counting. The radioactivity associated with the substrate 24,25(OH)2D3was estimated by liquid scintilla tion counting (44). Results were calculated as picomoles synthesized/hour/ incubation
and then standardized
Statistics.
for cell numbers.
All experiments
were repeated three times. Results were cx
pressed as mean ±SE relative to controls from untreated cells. All values of
X g, 4°C, 15 mm). The supernatant
gel, pH 6.8;
10% resolving
buffer containing
containing
gel, pH 8.8) at 200 V for 40 mm in
25 mM Tris, 192 mt@iglycine,
analysis
of data
program
(GraphPad
was
were analyzed
performed
Software,
using
San Diego,
by ordinary
the
Instat
version
CA). Group
or repeated
ANOVA.
2.04a
and multiple When
computer compar
statistical
dif
ferences were obtained, they were analyzed further by Tukey-Kramer multiple comparison tests. For single treatment comparisons, data were analyzed using an unpaired, two-tailed Student's t test. The statistics quoted in the text for multiple comparisons represent ANOVA, and those in the figure legends are the values derived from Tukey-Kramer multiple-comparison tests. RESULTS
proteins
nuclear extracts was quantified, aliquoted, and stored at —80°C. Protein Electrophoresis and Western Blotting. Nuclearproteins(5 @g/ lane) were diluted 1:2 in sample buffer [4% (w/v) SDS, 20% (v/v) glycerol, 0.1% (w/v) Dli', 125 msi Tris-HCI (pH 6.8), and 0.005% (w/v) bromophenol blue dye], heated at 100°Cfor 3 mm, and resolved by SDS-PAGE (4.5% electrophoresis
Analysis.
and
(4.6 X 25 cm; DuPont Co., Stevenage, United Kingdom) developed with a mobile phase of n-hexane:propan-2-ol:methanol [110:3:3.4 (v:v:v)] at 2 ml! mm. For each analysis, eluent fractions were collected and mixed with 1.5 ml aqueous scintillation fluid, and the radioactivity associated with labeled vita mm D3 metabolites was counted. Standards of metabolites of vitamin D3 [25(OH)D3,24,25(OH)2D3,la,25(OH)2D3, and 1,25(OH)2D2]or their tritiated
isons
‘
resuspended in 4 ml STM [0.25 M sucrose, 20 mist Tris-HC1, and 1.1 mrsi MgC12(pH 7.85)1 containing 0.5 misi PMSF (Sigma). Cells were pelleted
suspension
HPLC
(Amersham)
mRNA expression were corrected for expression of f3-actin mRNA. Statistical
United Kingdom). in 175-cm2
[26,27-3H]25(OH)D3
and 0.1% (wlv)
SDS (Mini-protean II system and reagents; Bio-Rad Laboratories, Richmond, CA). Proteins were electroblotted (100 V. 1 h, 4°C)to Immobilon P mem branes (Millipore, Watford, United Kingdom) in transfer buffer [25 mrviTnt, 624
Growth Assessments. There was a dose-dependent reduction in cell counts and [3H]thymidine uptake following treatment of HT-29 cells with l,25(OH)2D3 for 72 h (P < 0.0001; Fig. 1, a and b). 1,25(OH)2D3 produced a significant antiproliferative effect at concen trations of 10 I1 M. Treatment with 9-cis-RA alone did not result in a significant increase in cell numbers but caused a 40% increase in [3H]thymidine uptake at higher concentrations (lO@—bO@ M; P < 0.0001; Fig. 1, a and b). Cotreatment of HT-29 cells with l,25(OH)2D3 (10 13_l0_7 M) and increasing concentrations of 9-cis RA (10 IO_lO_7 M) resulted in inhibition of the antiproliferative
@
110
@f
ANTIPROLIFERATIVEACTIONS OF VITAMIN D3 AND 9-cis-RA
a
D3
9-cis RA 160
110
150
100 0
0
C
0 0
H
90
0
80
.@
0
.
.@ 0. 0
***
70
@0
!
120
E
E
@
H
0.
>,
110
.@. 60 C,)
C,)
50
100
40
90 control lxlO-13 lxlO-11 lxlO-9 lxlO-7
control lxlO-13 lxlO-11 lxlO-9 lxlO-7
Dose(M)
b
Dose (M)
D3
9-cis RA
120 H
120
110
@
@ @
Fig. 1. Growth studies of HT-29 cellt in response to 72-h treatment with 1,25(OH)2D3 and 9-cis-RA assessed by [3H]thy midine uptake (a) and cell counts (b), and growth studies of Caco-2 cells in response to 72-h treatment with l,25(OH)2D3 and 9-cis-RA assessed by l3Hlthymidine uptake (c). Results were standardized and expressed as mean percentage ± SE of the control ([3Hlthymidine, n = 4; cell counts, n 8). *, P < 0.05; **,
@
0 5SS• S
60
C) I
@70
A
***
I
60
50 control
10-13
10-11
10-9
control
10-7
10-13
Dose(M)
10-11
10-9
10-7
Dose(M)
C 110 @
100
H @@H-\
0
E 0
@
080
@
@S “ S
I 70
@
\@@* **A*
a)
@
-@
@
E
@
:@
@
@
Co
@
\@_
60
\@
@I
*4
I
“@** *** ***@@
@50
A
SR ***
j
40
‘@ - ---@
control 10-11 10-10 10-9 10-8 10-7
Dose(M) Fig. 2. Growth studies of HT-29 cells following 72-h cotreatment with l,25(OH)2D3 and 9-cis-RA assessed by [3Hjthymidine uptake (a) and cell counts (b), and growth studies ofCaco-2 cells following 72-h cotreatment with l,25(OH)2D3 and 9-cis-RA as assessed by [3H]thymidine uptake (c). U, 1,25(OH)2D3 treatment alone; •, l,25(OH)2D3 cotreated with 1 X l0@0 M 9-cis-RA; A, l,25(OH)2D3 cotreated with 1 X lO@ M 9-cis-RA; 0, l,25(OH)2D3 cotreated with I X 10_s M 9-cis-RA; and D, l,25(OH)2D3 cotreated with 1 X lO@ M 9-cis-RA.
Antagonism
of l.25(OH)2D3
action
is seen at 72 h when
cells
are cotreated
with
increasing
concentrations
of 9-cis-RA.
Complete
inhibition
of l,25(OH)2D3
action
is seen
when cells are cotreated with iO@ M 9-cis-RA. Results are expressed as mean percentage of the control. SE was < 10% for all results (data not shown; [3H]thymidine, a = 4; cell counts, n = 8). *, P < 0.05; @, P < 0.01; ***, P < 0.001 (Tukey-Kramer multiple-comparison tests relative to control). Data shown are derived from a single experiment, which was repeated three times with similar results.
626
ANTIPROLIFERATIVEACI'IONS OF
@lTAMIN D3 AND 9-cis-RA
b,@=
a
t-@
..•U@
•@
‘J
I@,
VDR
.@
‘J
•,@—.@
. . 0
S—
VDR @-4.4kB
0 RXRa
@ @
@—4
RXRa
. . a a RXR'y
I
4.4kB 9.5kB
: 7.5kB
RXRy
t•t@ @4.4kB
*—4.4kB
-9.5kB
@$ -4.4 kB
- 2.4kB
@—actin
@-actin
Fig. 3. Expression of VDR, RXRa, RXR'y, and f3-actin mRNAs in Northern blot analyses of polyadenylated RNA extracted from preconfluent HT-29 (a) and Caco-2 (b) cells following 6-h treatment with vehicle, l,25(OH)2D3 (1 X l0@—l X l0@ M), or 9-cis-RA (1 X 10°—iX l0@ M). Both cell lines expressed 4.6-kb mRNAs encoding VDR, a 5.5-kb
mRNA encoding RXRa, and 7- and 3.5-kb mRNA transcripts encoding RXR'y. RXRy cRNA cross-hybridizes to RXRa ( 0.05; Fig. 2, a response curve in the presence of any of the 9-cis-RA cotreatment and b]. concentrations, indicating a lack of synergy between the compounds (all Treatment of Caco-2 cells with l,25(OH)2D3 or 9-cis-RA resulted in a cotreatment concentrations, P < 0.0001; Fig. 2c). significant antiproliferative effect at concentrations of 10 lo_l07 M Receptor mRNA Expression. VDR (4.6 kb) and RXRa (5.5 kb) [l,25(OH)2D3, P < 0.0001; 9-cis-RA, P < 0.0001; Fig. id. In contrast mRNAs were expressed in HT-29 and Caco-2 cell lines. RXR@ to the blockade seen in HT-29 cells, cotreatment of Caco-2 cells with mRNA was not expressed in either cell line. Two specific RXRy l,25(OH)2D3 (b0 ‘ ‘—lO@ M) and increasing concentrations of 9-cis-RA mRNAs of 7 and 3.5 kb were identified in both cell lines. The pattern (10 ‘°—lO@ M) augmented antiproliferative effects; both hormones of VDR and RXR mRNA expression in HT-29 and Caco-2 cells was caused an approximate 50% reduction in [3H]thymidine uptake at identical to that identified previously in human colonic tumors and 1 X lO@ M when used individually, but there was a 75% reduction paired normal mucosa. The relative expression of VDR, RXRa, and following treatment with both agents at this concentration (data not RXRy mRNAs was not altered in either cell line following treatment 627
@1@
ANTIPROLIFERATIVEACTIONS OF VITAMIN D3 AND 9-cis-RA
1D31lxM
-
lOb
Caco@2
Western Analysis of VDR. A Mr 52,000 full-length VDR pro
@-9 108 iO-7
@S
tein was expressed in both cell lines (Fig. 4). In Caco-2 cells VDR protein levels increased following treatment with 1,25(OH)2D3 in a dose-dependent manner to a maximum of 25-fold at lO@ M l,25(OH)2D3 compared with control (P —0.0005; Fig. 4). In HT-29 cells, however, VDR protein expression was not affected by 1,25(OH)2D3 treatment (P = 0.4 1; Fig. 4). Treatment of either cell line with 9-cis-RA did not alter expression of the VDR protein (data not shown).
@-69kD
,1
-46kD
24-Hydroxylase mRNA Expression. There was very low basal
HT—29
,
I
@.‘i'
-69kD
‘@•“‘@
i
expression of 24-hydroxylase mRNA in HT-29 cells. However, 24hydroxylase mRNA expression increased in a dose-dependent manner following a 6-h treatment with 1,25(OH)2D3. Maximal induction was seen at lO@ M (P < 0.0001). Treatment with l0@ M 9-cis-RA resulted in a modest induction of 24-hydroxylase mRNA (control versus iO-@ M 9-cis-RA, P = 0.01; Figs. 5 and 6a). Cotreatment with 10 ‘ ‘ M l,25(OH)2D3 and bO@ M 9-cis-RA resulted in an additive induction of 24-hydroxylase mRNA [10 ‘ ‘s1 l,25(OH)2D3 versus b0 ‘M 1,25(OH)2D3 plus iO@ M 9-cis-RA, P = 0.03; Fig. 6b]. However, treatment with l0@ M l,25(OH)2D3 in combination with l0@ M 9-cis-RA resulted in synergistic interaction between both agents; there was a 2-fold induction of 24-hydroxylase relative to treatment with b,25(OH)2D3 alone (P = 0.001). Maximal induction
-46kD
Fig. 4. Exprestion of VDR protein were prepared from cells treated with and analyzed by Westem blotting (5 experiment was repeated three times formed. Both cell lines expressed
in HT-29 and Caco-2 cell lines. Nuclear proteins vehicle or l,25(OH)2D3 (1 X b_b_I X l0@ M) @g)using a monoclonal antibody against VDR. The with similar results, and densitometry was per M, 52,000 proteins, which were regulated by
l,25(OH),D3 in Caco-2 cells only.
@ with 1,25(OH)2D3 or 9-cis-RA for 6 h (Fig 3) or 24 h (not shown; P > 0.05 for all receptor mRNAs at both treatment times). RXR@ mRNA was not induced following treatment of HT-29 or Caco-2 cells with any agent.
HT—29 Treatment lxM
9-cis RA — i@-'@ iOn
i09
iO@
— iO―@ 10― i09
lO@
24(OH)ase 28S
Fig. 5. Expression of l,25(OH),D3 24-hydrox
.-
ylase mRNA in HT-29 and Caco-2 cells. Total
3.4kB
18S
RNA (15 jsgllane) prepared from cells treated for 6 h with vehicle, 1,25(OH)2D3 ( I X l0 13_I X l0'@ M),
or
9-cis-RA
(1
X
l0
‘i—iX
l0@
si)
was
subjected to Northern analysis. @3-Actin mRNA was used as an intemal control. Autoradiograph
expo
t3-actin
sure times were: 24-hydroxylase, 24 h in HT-29 cells and 48 h in Caco-2 cells; and @3-actin,16 h.
The experiment was repeated three times with tim ilar results, densitometry was performed, and the results were corrected for loading. A 3.4-kb 24hydroxylase mRNA was induced in both cell lines following treatment with l,25(OH)2D3 (I x 10 ‘ M in HT-29
cells
and
I X
10°
M in Caco-2
Caco-2
cells),
but only HT-29 cells demonstrated minor induction following treatment with 9-cis-RA (1 x l0@ M).
In addition, HT-29 cells express a minor 6-kb
Treatment
D3
9-cis RA
mRNA species at l0@ M.
lxM
-
i@-'@10-Il iO@ lO@ iO@
-
iO-'@ 10-― iO@
10
IO@
24(OH)ase 28S
34kB
185
@
@3-actin
• 0
• S 628
•-@ •.
.-S0
•
@ @ @
‘@
‘-
‘@
‘@
ANTIPROLIFERATIVEACTIONS OF
a
80
Caco-2 100
a D3 D
,@
@!TAMIN D3 AND 9-cis-RA
HT-29 100 -
=
g
‘@
9-cisRA
.
03
0
9-cisRA
80
-
0 (0 (I,
(0 0 U) 0,
0.
(0 a)
(0
0.
(I) to
0
S S U)
@;40-
to
0
a) > to a)
@
c%J S
20 -
.@
20
-
to S
@
Fig. 6. Graphs demonstrating hormone respon siveness of 24-hydroxylase mRNA expression in HT-29 and Caco-2 cells following 6-h treatment with l,25(OH)2D3 or 9-cis-RA (a) and I ,25(OH)2D3 or 1,25(OH)2D3 cotreated with
@
I X l0@ M 9-cis-RA (b). Hormone-induced
@
@-I1
@:z,
I
0-
I
.@
-=,
I
@2
;:
0-
0?
1*..
Q
(a
3—5)
and
are
relative
to
maximal
;: 6
g
changes in mRNA expression are shown graphi cally at mean hormone induction ratios ± SE
b
,
,@
6
9
‘@,@ ,@s
Dose of steroid (MJ
Dose of steroid (M)
induction
seen in HT-29 cells treated with 1 X l0@ M l,25(OH)2D3. All values were corrected for cx pression of the /3-actin intemal control. In a, sig
@5 .@
HT-29
Caco-2
nificance values are shown for Tukey-Kramer mul
@
@
tiple-comparison tests relative to controls. In b. significance values are shown for an unpaired, two-tailed Students t test comparing l,25(OH)2D3 treatment alone with I ,25(OH)2D3 plus cotreat ment of I x l0@ M 9-cis-RA for each concentra tion of l,25(OH),D1. *, P < 0.05; **, P < 0.01; ***,
>
a)
@ @
20 -
@.I-@J@x1@-I1 20
0-
0 .@
@
@ @
@2
6
;@
9)
6
was seen following treatment with l0@ M 1,25(OH)2D3; cotreatment with l0@ M 9-cis-RA did not enhance this maximal response (P = 0.65; Fig. 6b). Concentrations of 9-cis-RA of less than lO@ M did not affect the 24-hydroxylase mRNA response to l,25(OH)2D3. There was no detectable basal expression of 24-hydroxylase mRNA in Caco-2 cells. Treatment with b,25(OH)2D3 induced 24-hydroxylase mRNA in Caco-2 cells to only 20% of the maximal response evident in HT-29 cells. Induction was seen only after treatment with higher concentrations of 1,25(OH)2D3 (1 X 1O_8_1 x iO@ M; P < 0.0001; Fig. 5). In these cells, 9-cis-RA did not affect 24-hydroxylase mRNA expression when used either alone or as a cotreatment with (P = 0.2; Fig. 6, a and b).
r@.
9
Dose03 (M)
b,25(OH)2D3
40
to S
to
.@
@:
f
@,
c;
,,@
999
Dose 03 (M)
24-Hydroxylase Activity. HT-29 cells synthesized low basal 1ev els of 24,25(OH)2D3 (0.26 ±0.56 pmol/h/106 cells). Treatment of HT-29 cells with b,25(OH)2D3 resulted in increased synthesis of 24,25(OH)2D3; a maximal response was evident at l0@ M (28.45 ±0.76 pmol/h/l06 cells; P < 0.0001 ; Fig. 7a). Cotreatment with 1 X l0—@ M 9-cis-RA resulted in a synergistic interaction with b,25(OH)2D3 (l0@ M) to produce a 3-fold enhancement of
24,25(OH)2D3 synthesis relative to treatment with l0@
M
1,25(OH)2D3 (4.99 ±0.37 versus 1.63 ±0.48 pmoi/h1106 cells; P < 0.05; Fig. 7a). Treatment with 9-cis-RA alone did not affect 24,25(OH)2D3 synthesis (P > 0.05). There was no detectable basal synthesis of 24,25(OH)2D3 in Caco-2 629
ANTIPROLIFERATIVEACTiONS OF
@@FAMlN D3 AND 9-cis-RA
HT-29 @
Fig. 7. Graph demonstrating 24,25(OH)2D3 synthesis in HT-29 and Caco-2 cells following 24-h treatment with l,25(OH)2D3 or l,25(OH)2D3 cotreated with I x l0@ M 9-cis-RA. Hormone
U)
03
0
0
C)
30 -
***
30 -
I 03
N
E
induced changes in 24,25(OH)2D3 synthesis are shown graphically as mean values ±SE (n 3)
E
and are relative to maximal induction seen in HT-29cells treated with I X l0@ M 1,25(OH)2D3. For each experiment, rates of synthesis were cor rected for cell numbers, which were determined in parallel cultures using a hemocytometer after trypsinization. HT-29 cells showed very low basal levels of 24,25(OH)2D1 synthesis, which increased
0
-@ 0
0.
0.
]
D3
+ lxlO-7M
9-cis
RA
@
l0@—l
x
03 to
@20-
0
C >5 03 C,)
l0-@ M). Cotreatment with
I X I0@ M 9-cis-RA causes synergistic activation of 24-hydroxylase at l,25(OH)2D3 concentrations of I X l0° M. Caco-2 cells have no detectable basal levels of 24,25(OH)2D3 synthesis and show minor induction only in response to I X l0@ M l,25(OH)2D3, and cotreatment with I X l0@ M 9-cis-RA had no effect. Significance values are shown for Tukey-Kramer multiple-comparison tests relative to controls (, P < 0.05; P < 0.001). In addition, significance values for an unpaired, two-tailed Students t test comparing 1,25(OH)2D3 treatment alone with 1,25(OH)2D1 plus cotreatment of 1 X l0'@ si 9-cis-RA were
calculated (#, P < 0.05).
0
C
E
@11o@ >5
0 >5
.@ I0
C@1
—J-I-1II@i--=-1
csJ
control lxlO-11 lxlO-9
lxlO-7
control lxlO-11lxlO-9 lxlO-7
Dose (M)
cells. Following treatment with l0@ M l,25(OH)2D3, low levels of synthesis were detectable, but this did not reach significance (1.16 ±0.3 pmol/h/106 cells; P = 0.0871; Fig. 7b). Cotreatment with I X iO-@ M 9-cis-RA did not modify the response to l,25(OH)2D3 (P = 1.0).
DISCUSSION
@
D3
n 03 + lxlO-7M9-cis RA
E
E
following 24 h treatment with l,25(OH)2D3 (1 X
Caco-2
The human colon cancer cell lines HT-29 and Caco-2 express identical VDR and RXR mRNAs to human colon tissue and have been used widely as models of colonic cancer. Furthermore, treatment of HT-29 and Caco-2 cells with l,25(OH)2D3 has been shown to pro mote cell differentiation (13—17).In these studies, the antiproliferative responses to treatment with l,25(OH)2D3 were the same in both cell lines, but responses to 9-cis-RA differed. Experiments were per formed in media supplemented with CSS, which enabled us to study the interactions between l,25(OH)2D3 and retinoids without interfer ence by endogenous hormones. In this system, antiproliferative re sponses to 1,25(OH)2D3 ( l0 ‘—lO @° M) were appropriate for the binding affinity of VDR [dissociation constant for l,25(OH)2D3, 0.1
Dose (N)
variety of target cells (22—26),including colonocytes. Numerous in vitro studies demonstrate that nuclear receptor signaling is determined by a complex equilibrium within the nucleus; factors include the availability of ligand, the stoichiometry of heterodimer partners, the individual target gene activated, and other cell-specific proteins (5, 20, 22—25,46—49).In light of this, several factors could be implicated in the divergent responses of HT-29 and Caco-2 cells to 9-cis-RA. A primary factor could be the cell-specific regulation of VDR and RXR proteins. Previous data, derived from several colonic cell lines and a variety of methods, concerning the regulation of VDR by l,25(OH)2D3 have been conflicting (14—16).Our Northern analyses demonstrated that l,25(OH)2D3 and 9-cis-RA do not regulate VDR or RXR
mRNA
expression
in either
cell line. However,
l,25(OH)2D3
increased VDR protein in Caco-2 cells only, implying posttranscrip tional regulation of the receptor; posttranslational stabilization of the VDR protein by l,25(OH)2D3, in the absence of transcriptional reg ulation, has been demonstrated before in cell lines derived from other tissues (50). In contrast, 9-cis-RA did not regulate the VDR protein in either cell line, indicating that regulation of VDR does not account for the divergent responses to this agent. However, we have not studied aM; Ref. 39]. These effects were blocked by 9-cis-RA in HT-29 cells RXR protein expression and cannot exclude its regulation as a deter but potentiated by 9-cis-RA in Caco-2 cells, implying that the actions minant of cell-specific responses to 9-cis-RA. of 9-cis-RA may be cell specific. Alternatively, 9-cis-RA could influence cellular responses to l,25(OH)2D3 is implicated as a protective agent against the devel l,25(OH)2D3 by regulation of 24-hydroxylase expression. 24-Hy opment of colorectal cancer in man (6, 7), and our data support its droxylase promotes the catabolism of l,25(OH)2D3, and its induction chemotherapeutic potential. However, the molecular mechanisms Un by l,25(OH)2D3 has been reported previously in HT-29 and Caco-2 denying l,25(OH)2D3 action in the large bowel have not been studied. 9-cis modifies l,25(OH)2D3 action in vitro in severalcell types (24, cells (15, 51). Here, we report l,25(OH)2D3-mediated induction of 24-hydroxylase mRNA in both cell lines but enzyme activity in only 45—47), but this is the first demonstration that it modulates HT-29 cells. In contrast, 9-cis-RA induced 24-hydroxylase mRNA 1,25(OH)2D3 action in the colon. The cell-specific responses of and enzyme activity in HT-29 cells but not in Caco-2 cells. We HT-29 and Caco-2 cells to 9-cis-RA caution against the use of these cell lines as representative predictors of antiproliferative response to propose that this induction of 24-hydroxylase enhances the catabolism l,25(OH)2D3. Furthermore,our dataraise doubtregardingthe poten of l,25(OH)2D3 to explain in part why 9-cis-RA blocked 1,25(OH)2D3 antiproliferative actions only in HT-29 cells. This mech tial use of 9-cis-RA in colorectal cancer. Functional heterodimerization between VDR and RXR may play a anism may be particularly important at physiologically active concen ‘—l0@ M), because the addition of central role to control the specificity of l,25(OH)2D3 signaling in a trations of l,25(OH)2D3 (10 ‘ 630
ANTIPROLIFERATIVE
ACTIONS OF VITAMIN D3 AND 9-cis-RA
9-cis-RA doubled the activity of 24-hydroxylase at these concentra
6. Garland,C., Shekdlle,R. B., Barrett-Connor, E., Crique,R. B., Rossof,A. H., and
tions of 1,25(OH)2D3. These studies provide some insight into the cell-specific actions of 1,25(OH)2D3 and 9-cis-RA in human colon cancer cells, but several other mechanisms must be considered. It is possible that activation or metabolism of retinoids differs between HT-29 and Caco-2 cells. No studies exist concerning retinoid metabolism in colonocytes, although it is known to be cell specific and under complex hormonal control (52). Furthermore, the precise details by which VDR and RXRs interact with the general transcription apparatus to regulate target gene expression are unknown. Recent findings concerning other nuclear receptors may be applicable; a new family of transcription factors which interact with T3Rs has been identified (53, 54), and similar proteins associate with the estrogen receptor (55). These coactivator proteins interact with both the hormone receptor and the general transcription apparatus but have restricted tissue expression. More recently, corepressor proteins have been identified, which may mcdi ate cell-specific repression of target gene expression induced by VDR and other nuclear receptors (56). By analogy, similar proteins that modify VDR or RXR activity could account for the cell-specific responses described in this report. In addition, other receptors, includ ing T3Rs and RARs, can heterodimerize with VDR and may influence ligand-dependent transcription (48, 57). In this context, study of the 24-hydroxylase promoter in transfection and footprinting studies should provide insight into the cell-specific responses described in HT-29 and Caco-2 cells. The ultimate response to l,25(OH)2D3 and 9-cis PA in the colon will be dependent on the individual cell and its nuclear receptor, together with the relative concentrations of ligands and their metabolites. These corn plex issues indicate that the use of HT-29 and Caco-2 cells as represent ative models of colon cancer is too simplistic. Based on our findings, we suggest that human colorectal carcinomas may display varying responses to vitamin D and retinoids, which are difficult to predict and cannot be anticipated by hormone receptor status.
Paul, 0. Dietary vitamin D and calcium and risk if colorectal cancer: a 19-year prospective study in men. Lancet, 1: 307—309.1985.
ACKNOWLEDGMENTS HPLC analysis was performed in the University of Manchester Department of Medicine, Manchester Royal Infirmary, with the cooperation of Dr. M. E. Hayes and S. Heys. Metabolite standards for HPLC were provided by Prof. E. B. Mawer. Dr. Bob Cowan (University of Glasgow, Glasgow, United Kingdom)
performed
the l,25(OH)2D3
assays
on untreated
FCS and CSS. Dr.
Lise Binderup (Leo Pharmaceutical Products, Ltd., Ballerup, Denmark) pro vided
the 1,25(OH)2D3,
and Dr. David
Moore
(Massachusetts
General
7. Garland, C. F., Comstock, G. W., Garland, F. C., Felsing, K., Shaw, E. K., and
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(Lond.), 9:
187—190, 1988.
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Hos
pital, Boston, MA) provided the 9-cis-RA. The VDR cDNA was a gift from Dr. Bert O'Malley (Baylor University, Houston, TX), the RXRa, RXR@,and RXRy cDNAs were donated by Dr. Ron Evans (Salk Institute, San Diego, CA), and the human 24-hydroxylase cDNA was provided by Dr. H. DeLuca
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X receptor hetcrodimer-mediated
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