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Tumor Biology |
Cancer Research Campaign Colorectal Tumour Biology Research Group, Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom [G. D. D., C. P.]; University Department of Surgery, Bristol Royal Infirmary, Bristol BS2 8HW, United Kingdom [M. G. T.]; Biological Research and Development, LEO Pharmaceutical Products, DK-2750 Ballerup, Denmark [L. B.]; and Department of Oral and Dental Science, University of Bristol Dental School, Bristol BS1 2LY, United Kingdom [A. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSMeasurement of Differentiation:...RESULTSDISCUSSIONREFERENCES |
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,25-dihydroxyvitamin
D3, has previously been shown to inhibit growth and promote
differentiation of colon cancer cells. The vitamin D analogue, EB1089,
is currently under clinical trial in a variety of cancers because of
its growth-inhibitory effects in vitro and reduced
hypercalcemic effects in vivo. The mechanism of growth
inhibition by EB1089, however, remained to be determined. In this study
we examined the effects of 1,25-dihydroxyvitamin D3 and
EB1089 on five colorectal tumor cell lines (two adenoma and three
carcinoma) to determine the mechanism of growth inhibition and to
ascertain whether premalignant adenoma cells were responsive to these
agents. 1,25-Dihydroxyvitamin D3 and EB1089 induced
p53-independent apoptosis in adenoma and carcinoma cell lines in a
dose-dependent manner between 10-10 and 10-6
M. EB1089, as well as inducing apoptosis, increased the
proportion of cells in the G1 phase, particularly in
the adenoma cell lines. In two of the three carcinoma cell lines (SW620
and PC/JW), levels of apoptosis induced by EB1089 were similar or
greater than those induced by 1,25-dihydroxyvitamin D3.
Although the carcinoma cell line HT29 was relatively resistant to
apoptosis induced by EB1089 compared with 1,25-dihydroxyvitamin
D3, EB1089 markedly inhibited cell yields. These
observations offer promise for the clinical use of EB1089. To determine
whether the induction of apoptosis by 1,25-dihydroxyvitamin
D3 and EB1089 was potentially via a differentiation
pathway, alkaline phosphatase activity was measured as a marker of
differentiation. Induction of alkaline phosphatase was observed in the
floating apoptotic cells as well as in the adherent population. A link
between the induction of differentiation and apoptosis by
1,25-dihydroxyvitamin D3 and EB1089 is suggested by the
occurrence of apoptosis subsequent to the induction of differentiation.
To investigate the molecular pathway to apoptosis induction, members of
the Bcl-2 family of proteins were examined (Bcl-2, Bcl-x, Bax, and
Bak). Decreased Bcl-2 was observed in some cell lines, particularly in
response to EB1089, but was not essential for apoptosis. Levels of the
proapoptotic protein Bak, however, were consistently increased in all
of the five cell lines in association with apoptosis induced by either
agent. The results implicate Bak protein in the induction of apoptosis
by 1,25-dihydroxyvitamin D3 or its analogue EB1089. The
ability of EB1089 to induce apoptosis in colorectal carcinoma cells
suggests that this or other vitamin D analogues may prove clinically
effective for the treatment of colorectal cancer. Furthermore, the fact
that it induces cell cycle arrest and apoptosis in the premalignant
adenoma cells may suggest an application in colorectal cancer
chemoprevention. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of Differentiation:...RESULTSDISCUSSIONREFERENCES |
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,25-dihydroxyvitamin D3, is growth inhibitory
to colonic adenocarcinoma cells, promoting cellular differentiation
(7, 8, 9)
. In familial adenomatous polyposis patients, it has
been demonstrated to inhibit proliferation of rectal adenomas
(9)
. 1,25-Dihydroxyvitamin D3 has
been reported to induce apoptosis in breast cancer cell lines
(10)
. Furthermore, there has been a brief report that
1,25-dihydroxyvitamin D3 induces apoptosis in
HT29 colon cancer cells (11)
, but the mechanisms of
apoptosis have not been determined, and there have been no
investigations into whether 1,25-dihydroxyvitamin
D3 induces apoptosis in premalignant colon cells.
These properties of 1,25-dihydroxyvitamin D3
could be responsible for the reported cancer preventive effects of the
vitamin, particularly if 1,25-dihydroxyvitamin
D3 induces apoptosis in premalignant adenoma
cells.
The growth-inhibitory properties of 1,25-dihydroxyvitamin
D3 could potentially be harnessed
therapeutically. However, dose-limiting effects on calcium metabolism
preclude its clinical use in the treatment of cancer. Emphasis has,
therefore, been placed on the development of structurally related
analogues that retain potent antiproliferative activity but have
reduced hypercalcemic effects. One such analogue is EB1089, which, in
breast cancer cells, has a 100-fold greater antiproliferative effect
than 1,25-dihydroxyvitamin D3
(12)
and has been shown to block cell cycle progression in
the G1 phase by means of p21 up-regulation
(13)
. In addition, EB1089 has been reported to induce
apoptosis in myeloma cells (14)
and in breast cancer cells
(15)
. The mechanisms of its growth-inhibitory properties
on colorectal cancer cells remain undefined. In particular, there have
been no reports of whether EB1089 induces apoptosis in colorectal tumor
cells.
1,25-Dihydroxyvitamin D3 behaves as a steroid
hormone and regulates gene transcription through a nuclear receptor,
the VDR,2
a member of the steroid/thyroid hormone receptor superfamily. The VDR
is a ligand-activated nuclear receptor that binds to specific DNA
binding sites known as VDREs to activate or repress the transcription
of target genes that regulate a variety of biological processes
(16)
. These VDREs may be direct repeats of hexameric
core-binding motifs separated by three nucleotides (DR3) or inverted
palindromic repeats separated by 9 nucleotides (IP9; Ref.
17
). EB1089 has been shown to preferentially target
IP9-type VDREs (12)
. The VDR is thought to act
preferentially in a heterodimeric complex with the retinoid X receptor
(RXR) but may also act as a homodimer (17)
or in
association with the RAR all-trans retinoic acid receptor
(16)
or the thyroid hormone receptor (T3R; Ref.
18
). The VDR is expressed in colonic epithelial cells
(19
, 20)
, which suggests that the colon is an important
target organ for vitamin D3 action. The VDR is
also expressed in colorectal cancer tissues irrespective of Dukes’
stage or degree of differentiation (21)
. Effects of
1,25-dihydroxyvitamin D3 mediated by the VDR
are referred to as the genomic actions of vitamin
D3. Additional effects, such as rapid increases
in intracellular calcium and induction of phospholipase A2 and protein
kinase C activities, known as the nongenomic effects of vitamin
D3, are proposed to be mediated by the action of
1,25-dihydroxyvitamin D3 on a putative plasma
membrane receptor (22)
.
The aim of this study was to determine the mechanisms of growth
inhibition by the active metabolite of vitamin D
(1,25-dihydroxyvitamin D3) and its analogue
EB1089 on colorectal adenoma as well as carcinoma cells, initially to
determine whether this involves cell cycle arrest, differentiation,
and/or apoptosis. Two premalignant adenoma cell lines and three
adenocarcinoma cell lines were used to determine whether adenoma cells
were responsive and to compare their response with that of carcinoma
cells, because chemopreventive agents may act at different stages of
the adenoma to carcinoma sequence. For example, salicylate (the active
metabolite of aspirin) is protective against colorectal cancer and
induces more extensive apoptosis in carcinoma cells than in adenoma
cells (23)
. In this paper, we demonstrate that
1,25-dihydroxyvitamin D3 and EB1089 induce
p53-independent apoptosis subsequent to the induction of the
differentiation marker, ALP, in all of the five colorectal tumor
cell lines. These observations may, in part, explain the
epidemiological link between vitamin D and a reduced risk for
colorectal cancer and offer promise for the use of vitamin D analogues
such as EB1089 for the treatment or prevention of colorectal cancer.
Furthermore, we show that elevated Bak levels may provide a molecular
basis for the induction of apoptosis by 1,25-dihydroxyvitamin
D3 or EB1089.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of Differentiation:...RESULTSDISCUSSIONREFERENCES |
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Treatment with 1,25-Dihydroxyvitamin D3 or EB1089
1,25-Dihydroxyvitamin D3 and
EB1089 were synthesized in the Department of Chemical Research (Leo
Pharmaceuticals, Ballerup, Denmark). The compounds were dissolved in
propan-2-ol at 4 mM. Dilution was performed in ethanol to
give stock solutions of 2 x 10-4
M and aliquots of the stock solution were stored at
-20°C. All of the solutions containing 1,25-dihydroxyvitamin
D3 and EB1089 were protected from the light. Cells were
seeded at 1 x 106 cells per T25 flask for
HT29, SW620, PC/JW and RG/C2, and at 2 x 106 cells per T25
flask for the AA/C1 cell line. These seeding densities yielded a
similar number of cells at the day of treatment 3 days later. At the
time of treatment, the cells were in the exponential growth phase.
Cultures were treated with either 1,25-dihydroxyvitamin
D3 or EB1089 at concentrations ranging from
10-10 M to 10-6 M in
triplicate for harvests at each of three time points.
Solvent-containing culture medium was used to treat the control flasks
and to perform the serial dilutions, such that the same volume of
solvent was present in all of the treatment conditions (0.5%).
Preliminary experiments demonstrated that medium changing every day
with fresh 1,25-dihydroxyvitamin D3 resulted in a
greater reduction in the adherent-cell yield. Therefore, for all of the
experiments described in this paper, the medium was changed every day
once treatment commenced. At each medium change, the cells floating in
the medium were collected and counted using a counting chamber.
Adherent-cell counts were obtained after 2, 4, and 7 days of treatment.
Identification of Apoptosis
Acridine orange/ethidium bromide dual staining was used to
identify apoptotic cells by staining the condensed chromatin. Unfixed
cells were stained with 5 µg/ml acridine orange together with 5
µg/ml ethidium bromide in PBS for 10 min and were then viewed by
fluorescence microscopy. Acridine orange stains early-stage apoptotic
cells. Only late-stage apoptotic cells that have lost membrane
permeability stain with ethidium bromide. Ethidium bromide is also
useful in that it identifies cells that are necrotic, because they have
lost membrane permeability but do not contain condensed chromatin. At
least 300 cells were scored for each sample for three independent
experiments.
To further demonstrate that the floating cells were apoptotic, 106 cells were used to prepare samples for DNA laddering by the method of Smith et al. (31) . As a positive control, mouse thymocytes, treated with 10-7 M dexamethasone for 16 h, were used. Cells were lysed and digested for 1 h at 50°C in 20 µl of a solution containing 5 mM EDTA, 25 mM Tris-HCl (pH 8.0), 5 mg/ml lauryl sarcosine, and 0.5 mg/ml proteinase K. RNase A (10 µl) from a stock solution of 0.5 mg/ml was added to the samples, which were incubated for an additional hour at 50°C. The samples were heated to 70°C, and 10 µl of melted loading buffer, containing 10 M EDTA, 1% (w/v) low-melting-point agarose, 40% (w/v) sucrose, and 0.25% bromphenol blue, were added to each sample. The samples were immediately dry-loaded into a gel of 2% agarose type II (Sigma) and 0.7% Infinity Agarose Enhancer (Oncor). To make up the gel, the agarose and enhancer were slurried in 7 ml of ethanol and then made up to 70 ml with Tris acetate buffer [40 mM Tris, 5 mM sodium acetate, and 1 mM EDTA (pH 8.1)] and supplemented with 0.5 µg/ml ethidium bromide. The gel was run at 40 V for 5 h in Tris-acetate buffer.
Cell Cycle Analysis
One million trypsinized adherent cells were washed in PBS, fixed
in 70% ethanol, and stored at -20°C for 1 week. In preparation for
cell cycle analysis, the cells were pelleted, resuspended in 1 ml PBS
containing 0.2 mg/ml propidium iodide (Sigma) and 0.15 µg/ml RNase
(Sigma), and incubated at 37°C for 30 min in the dark. Cells were
then left overnight at 4°C in the dark before analysis on a
fluorescent-activated cell sorter (Becton Dickinson). Cells were
excited with a single 488-nm argon laser, and the fluorescence was
detected through a 585 ± 22-nm filter. Linear
red-orange fluorescence (FL3) data were collected in list format to
10,000 total events, and the distribution of cells within the cell
cycle was estimated using a CellFIT software program, based on a
rectangular S-phase model.
SDS-PAGE Western Blotting
Sample Preparation for SDS-PAGE Western Blotting Detection of
Bcl-2 Family of Proteins.
Trypsinized adherent cells (106) were resuspended
in 50 µl of gel sample buffer [0.125
M Tris-HCl (AnalaR, BDH; pH6.8), 20%
(v/v) glycerol, 2% (w/v) SDS, 10% (v/v) ß-mercaptoethanol, and
0.25% (w/v) bromphenol blue], and the tubes were placed in a
boiling-water bath for 5 min. Samples were stored at -20°C before
electrophoresis.
SDS-PAGE.
Resolving gel was made up at 12.5% Protogel [30% (w/v) acrylamide,
0.8% (w/v) bisacrylamide; Bio-Rad] and stacking gel at 4.5%. Gels
were run in a buffer solution of 192 mM glycine (AnalaR,
BDH), 25 mM Tris (AnalaR, BDH), and 1% SDS and were then
electroblotted onto Immoblion polyvinylidine difluoride (PVDF) membrane
in a transfer buffer of 192 mM glycine (AnalaR, BDH), 10
mM Tris (AnalaR, BDH), and 20% (v/v) methanol (AnalaR,
BDH).
Immunoblotting for the Bcl-2 Family of Proteins.
Antibodies used were as follows: (a) Bcl-2 mouse
monoclonal clone 124 (kindly provided as culture supernatant by David
Mason, University of Oxford, United Kingdom), used at a dilution of
1:100; (b) Bax N20 rabbit polyclonal (Santa Cruz), used at a
dilution of 1:500; (c) Bak G23 rabbit polyclonal (Santa
Cruz) used at a dilution of 1:500;(d) Bak monoclonal clone
G317–2 (PharMingen) used at a dilution of 1:1000; and (e)
Bcl-x L19 rabbit polyclonal (Santa Cruz) used at a dilution of 1:500. A
mouse monoclonal -tubulin antibody (Sigma) was used to assess equal
loading of the gels at 1:1000. Membranes were blocked in milk block
buffer [10 mM Tris/HCl (pH 7.4), 150
mM NaCl, 4% nonfat dried milk] for 1 h or
overnight at 4°C. Incubations in primary antibody were conducted for
4 h at room temperature or overnight at 4°C. Membranes were
rinsed in distilled water, washed twice for 10 min in milk-block buffer
and then twice for 10 min in Tween buffer [10 mM
Tris (pH 7.4), 150 mM NaCl, 0.2% Tween 20 (w/v);
Sigma], followed by an additional 10 min in milk-block buffer.
Secondary antibodies were antimouse or antirabbit horseradish
peroxidase-conjugated antibodies (Sigma). Membranes were incubated in
the appropriate secondary antibody at 1:1000 for 45 min at room
temperature. The membranes were then washed for 20 min in milk-block
buffer and for 10 min in Tween buffer. After rinsing in distilled
water, peroxidase activity was detected using an Enhanced
ChemiLuminescence (ECL) detection kit (Amersham) following the
manufacturers’ protocol.
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Measurement of Differentiation: The ALP Assay |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of Differentiation:...RESULTSDISCUSSIONREFERENCES |
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,25-dihydroxyvitamin D3 or EB1089 on colonic
cell differentiation. ALP activity has previously been used as a
measure of colonic cell differentiation in response to butyrate and was
used to demonstrate that butyrate induces apoptosis by a
differentiation-dependent pathway in colonic tumor cells (32
, 33)
. Triplicate flasks were set up to harvest for the assay in
parallel with the flasks for the cell counts, and the cultures were
treated as described above. Medium was removed from the flasks, which
were each washed with 2.5 ml of 0.15
M NaCl and then incubated for 5 min
at 37°C in 0.25% sodium deoxycholate (Sigma). Cells were removed
from the flasks using a cell scraper, and the lysate was centrifuged at
4000 rpm for 5 min. For measurement of ALP activity, an ALP reagent kit
(Sigma) was used following the method of Bowers and McComb
(34)
. Reaction mixture (1 ml) consisting of 7
mM p-nitrophenyl phosphate, 0.1
M sodium bicarbonate, and 5
mM MgCl2 was added to
spectrophotometer cuvettes containing 20 µl of sample lysate. The
absorbance was measured at 410 nm using a UV-Unicam spectrophotometer
for 1 min at 30°C. Protein content for each sample was measured by
the Bradford colorimetric method (35)
with reference to
BSA standards (Bio-Rad). The absorbance was measured at 595 nm using an
UltroSpec III spectrophotometer (Pharmacia). The calculation of protein
content per sample allowed the ALP activity to be expressed as units
per µg of protein. |
RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of Differentiation:...RESULTSDISCUSSIONREFERENCES |
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,25-Dihydroxyvitamin D3 and Its Analogue EB1089 in
Colorectal Adenoma and Carcinoma Cell Lines.,25-dihydroxyvitamin
D3 and EB1089 induced growth inhibition and cell
shedding into the medium. We have previously demonstrated that the
majority of colon cells that are shed into the medium, either
spontaneously or by treatment with agents such as butyrate or radiation
(36
, 37)
, are apoptotic. Because of rapid detachment of
cells entering apoptosis, very few apoptotic cells were seen in the
adherent population. The proportion of cells floating in the medium
can, therefore, be used as a measure of the extent of apoptosis in the
cultures if the floating cells are confirmed to be morphologically
apoptotic. Furthermore, the floating cells produce a DNA ladder
indicative of apoptotic oligonucleosomal DNA cleavage. Adherent cells,
by contrast, rarely demonstrate this DNA cleavage pattern, because
oligonucleosomal fragmentation of DNA is a late event in apoptosis. To
determine whether the cells that were shed in response to
1,25-dihydroxyvitamin D3 or EB1089 represented
apoptosis induced by the treatment, the adherent and floating cells
were separately examined for apoptotic morphology using acridine
orange/ethidium bromide dual staining. At each time point (2, 4, and 7
days of treatment) and for each agent, the floating cells collected
from the treated cultures had a greater proportion of cells with the
condensed chromatin pattern definitive of apoptosis (38)
than the control cultures (Fig. 1A)
. In each case, there was a dose-dependent increase in the
apoptotic index of the floating cells. This was true even for the top
concentration of 10-6 M,
which indicated that the pronounced increases in the proportion of
cells that were floating in the medium of cultures receiving the top
concentration were attributable to the induction of apoptosis and not
attributable to necrosis. To further confirm this observation, DNA gel
electrophoresis was used to demonstrate DNA laddering indicative of
internucleosomal fragmentation in the floating cells (Fig. 1B)
. In the PC/JW carcinoma cell line, this was accompanied
by an increase in the apoptotic index of the adherent population that
was observed (a) by fluorescent microscopy (data not shown);
and (b) as a similar proportion of adherent cells having a
sub-G1 content on flow cytometric analysis (Fig. 1C)
. For example at the 7-day time point, the percentage of
adherent cells that were apoptotic was 8.5% in the cultures treated
with 10-6 M 1,25-dihydroxyvitamin
D3 compared with 1.5% in the control cultures. For the
other four cell lines, apoptosis in the form of chromatin condensation,
DNA laddering, or reduced DNA content was only seen in the floating
cell populations. For each cell line treated with each agent, the
floating cells were confirmed to be apoptotic by acridine
orange/ethidium bromide dual staining and by DNA laddering. Once we
were satisfied that the cells that were shed into the medium of the
treated cultures represented apoptosis, we used the proportion of cells
floating in the medium as a measure of the extent of apoptosis taking
place in the cultures, as we had done for previous studies of the
differentiation agent, sodium butyrate (36)
.
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,25-Dihydroxyvitamin D3 Treatment Reduces the
Attached Cell Yield and Induces Apoptosis.,25-dihydroxyvitamin D3 was
required for the optimal induction of apoptosis. The data shown in Fig. 2
are the results obtained after 7 days of treatment, and give an overall
effect of 1,25-dihydroxyvitamin D3 treatment
on adherent-cell yield and the extent of apoptotic cell shedding. The
proportion of cells shed into the medium was the cumulative number of
floating cells collected each day as a proportion of the total numbers
of cells at the time of adherent-cell harvest (attached cell yield plus
the total number of floating cells generated during the treatment
time). To enable the effects of 1,25-dihydroxyvitamin
D3 on the five different cell lines to be easily
compared, the adherent-cell yield is presented as a percentage of the
control yields, and the proportion of cells shed into the medium is
expressed as a fold control value. Individual (daily) floating cell
numbers and the adherent-cell yields for days 2 and 4 are not shown.
1,25-Dihydroxyvitamin D3 was growth inhibitory
and induced apoptosis in all of the five cell lines (Fig. 2)
. The
carcinoma cell lines HT29 and SW620 were highly sensitive to
1,25-dihydroxyvitamin D3-induced apoptosis at
10-6 M. Although apoptosis was
obtained after only 2 days of treatment in all of the five cell lines,
there was initially differential sensitivity to apoptosis with a
greater extent of apoptosis in the adenoma cell lines, and, therefore,
a longer treatment time (7 days) was required to demonstrate the extent
to which 1,25-dihydroxyvitamin D3 was able to
induce apoptosis in the carcinoma cell lines. By day 7 of treatment
with fresh vitamin applied each day, 1,25-dihydroxyvitamin
D3 induced apoptosis in each of the five cell
lines.
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,25-dihydroxyvitamin D3. EB1089 reduced
the adherent-cell yield (Fig. 3A)
and induced apoptosis in each cell line in a
dose-dependent manner, although HT29 was relatively resistant to
EB1089-induced apoptosis (Fig. 3B)
. Interestingly, as was
observed with 1,25-dihydroxyvitamin D3 at day
4, the adenoma cell lines were more sensitive than the carcinoma cell
lines to apoptosis induced by EB1089 early in the time course (data not
shown). The carcinoma lines varied in their apoptotic responses to
EB1089. By day 7 of treatment, HT29 and SW620 had comparable levels of
growth inhibition (Fig. 3A)
; however, SW620 demonstrated a
marked increase in apoptosis (>8-fold higher for the highest
concentration than for the control), whereas HT29 demonstrated
relatively low levels of apoptosis (<2-fold the control for the
highest concentration; Fig. 3B
). Thus, growth inhibition did
not always correlate with apoptosis.
|
,25-Dihydroxyvitamin D3.,25-dihydroxyvitamin D3
treatment, a G1 arrest was evident only with the
highest concentration (10-6 M) in
RG/C2, AA/C1, and PC/JW (Fig. 4A
shows data from day 7). The most sensitive cell line was
the adenoma cell line RG/C2, for which there was a progressive increase
in the proportion of cells in the G1 phase and a
reduction in the proportion of cells in the S phase and
G2-M from day 2 onwards (data not shown).
Inasmuch as a cell cycle arrest was only observed when using the
highest concentration of 1,25-dihydroxyvitamin
D3, this does not account for the growth
inhibition by 1,25-dihydroxyvitamin D3.
|
.
The carcinoma cell lines (including HT29, which was insensitive to the
induction of a cell cycle arrest with 1,25-dihydroxyvitamin
D3) showed an increase in
G1:S phase ratio but only with the top
concentration of EB1089. The G1:S phase ratio of
the carcinoma cell lines at the highest concentration of EB1089
(10-6 M) was lower than that of the adenoma
cell lines at 10-7 M.
In PC/JW, 1,25-dihydroxyvitamin D3 and
EB1089 induced a sub-G1 peak (example shown in
Fig. 1C
). This increased with both time and concentration in
this particular cell line and is indicative of apoptosis in the
adherent-cell population. The percentage of cells in the
sub-G1 peak was consistent with the percentage of
cells with apoptotic morphology (visualized in the propidium
iodide-stained cells under the fluorescence microscope). Because no
sub-G1 peak was observed in the adherent
populations of the other four cell lines after treatment with either
agent, this was not used to assess the extent of apoptosis in all of
the cultures.
1,25-Dihydroxyvitamin D3 and EB1089 Induce
Expression of the Proapoptotic Protein, Bak, in Association with the
Induction of Apoptosis.
Three experiments were performed for each of the five cell lines,
and, for these experiments, samples of the adherent cells were run for
Western blot analysis of the Bcl-2 family of proteins. The proteins
that we chose to investigate in this study were the antiapoptotic
proteins Bcl-2 and Bcl-xL and the proapoptotic
proteins Bax and Bak. The five cell lines had different basal levels of
Bcl-2 protein. RG/C2 and SW620 had high levels, PC/JW and HT29 had
moderate levels, and AA/C1 did not express detectable levels of Bcl-2.
All of the cell lines expressed similar levels of
Bcl-xL, Bax, and Bak. For all of the blots in
this study, protein loading was confirmed by reprobing for -tubulin.
The effects of the top concentration of 1,25-dihydroxyvitamin
D3 and EB1089 are summarized in Tables 1
and 2
,
respectively. The most consistent finding was that each of the five
cell lines showed increased levels of Bak in response to either
1,25-dihydroxyvitamin D3 or EB1089. In
response to 1,25-dihydroxyvitamin D3, the
increase in Bak protein was dose dependent in some cell lines (AA/C1
and HT29), and, in others, all of the concentrations up-regulated Bak
protein to an apparently equal extent (RG/C2 and SW620; Fig. 5A
). For PC/JW, a pronounced increase in Bak was detected with
the top concentration only. The HT29 cell line showed an increase in
Bak protein at days 4 and 7 only (i.e., relatively late),
paralleling the late induction of apoptosis by 1,25-dihydroxyvitamin
D3 in this cell line. In response to EB1089, Bak
levels were increased with all of the concentrations of EB1089 at all
of the time points in the adenoma and carcinoma cell lines (see Fig. 5A
for examples). For PC/JW, the increase in Bak was
detected at the day-2 time point, i.e., the increase
occurred earlier and at lower concentrations in response to EB1089
compared with 1,25-dihydroxyvitamin D3 (Fig. 5A)
.
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and 2)
. For example, Bcl-2 levels were reduced in response to EB1089 in the
RG/C2 and PC/JW cell lines but were increased in HT29 and SW620 (Fig. 5B)
. Bax expression was increased at some of the time points
after EB1089 treatment in AA/C1, RG/C2, and HT29 (Fig. 5B)
.
Changes in Bcl-xL expression were small and the
shorter proapoptotic form of Bcl-x, Bcl-xS, was
not detected in any of the cell lines in control or treated samples
(data not shown).
1,25-Dihydroxyvitamin D3 and EB1089 Induce ALP
Activity in Colorectal Tumor Cells.
ALP activity has previously been used as a measure of colonic
cell differentiation in response to butyrate and to demonstrate that
butyrate induces apoptosis by a differentiation-dependent pathway in
colonic tumor cells (32
, 33)
. In these studies, the
spontaneous floating cells had higher ALP activity than the adherent
cells (indicating that at least some of the spontaneous floating cells
are derived from a differentiated population), and treatment with the
differentiation agent, butyrate, induced ALP activity in adherent and
floating cell populations. To determine whether
1,25-dihydroxyvitamin D3 or EB1089 also
induced apoptosis via a differentiation pathway, ALP activity was
measured at days 4 and 7 of treatment as a reliable differentiation
marker for colonic epithelial cells. ALP activity was measured in
floating and adherent cells separately, so that we could detect not
only whether differentiation was induced in the adherent-cell culture
but also whether the floating apoptotic cells, induced by treatment
with 1,25-dihydroxyvitamin D3 or EB1089, had
higher ALP activity than the corresponding adherent-cell populations. A
higher ALP activity level would be consistent with the floating
apoptotic cells representing terminally differentiated cells that would
arise from the induction of differentiation in the adherent cells. In
the control cultures for each of the five cell lines, the ALP activity
was approximately 4-fold higher in the floating cells than in the
adherent cells, as has been shown in previous studies (32
, 33)
. In all of the five cell lines, 1,25-dihydroxyvitamin
D3-treated cultures had higher ALP activity in
both the adherent and floating cells, and the ALP activity increased
with increasing 1,25-dihydroxyvitamin D3
concentration (Fig. 6)
. It was noticeable that the cell line in which
1,25-dihydroxyvitamin D3 induced the least ALP
activity in the adherent-cell population was the HT29 carcinoma cell
line, although, as in the other cell lines, in the collected floating
cells of the treated cultures, there were greater ALP activities than
in those of the controls. Indeed, this finding is consistent with
observations by Tanaka et al. (8)
that very
little ALP activity was induced by 1,25-dihydroxyvitamin
D3 alone in HT29 cells. Similar patterns of ALP
induction were obtained using EB1089 (data not shown), which indicated
that, like 1,25-dihydroxyvitamin D3, the
analogue induces differentiation followed by apoptosis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of Differentiation:...RESULTSDISCUSSIONREFERENCES |
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One of the questions that we wished to address was whether other agents
known to induce differentiation in colonic epithelial cells would also
induce apoptosis in colonic tumor cells and whether this was via a
pathway of differentiation. We were interested in
1,25-dihydroxyvitamin D3 because this steroid
hormone had previously been shown to be growth inhibitory to colonic
tumor cells (9)
. A number of studies of the effects of
1,25-dihydroxyvitamin D3 on the colonic
carcinoma cell line HT29 have demonstrated the induction of
differentiation by the steroid (40
, 41)
or potentiation of
butyrate-induced differentiation (8)
. In the
hyperproliferative skin disease, psoriasis, keratinocytes are induced
to differentiate by 1,25-dihydroxyvitamin D3
and analogues (42)
, and vitamin D compounds form an
effective treatment. In mammary tumor cell lines,
1,25-dihydroxyvitamin D3 induces apoptosis
(10)
, whereas in the myeloid leukemia cell line, HL60, it
has been reported to confer resistance to apoptosis induced by calcium
ionophore and the chemotherapeutic drugs, arabinocytosine and etoposide
(43)
. These observations led us to question whether the
growth inhibition obtained in colonic carcinoma cells was attributable
to apoptosis and/or a cell cycle arrest. We also questioned whether
there was a link between differentiation and apoptosis and whether
premalignant adenoma cells would undergo apoptosis in response to
1,25-dihydroxyvitamin D3.
In all of the five cell lines (two adenoma and three carcinoma),
1,25-dihydroxyvitamin D3 induced growth
inhibition, the differentiation marker ALP, and apoptosis. The
apoptotic cells were shed into the medium. These cells were
morphologically apoptotic and demonstrated internucleosomal DNA
cleavage. In the PC/JW cell line, we have frequently noticed that, in
response to different agents, detachment during apoptosis is delayed
compared with the other four cell
lines3
and, in this cell line, 1,25-dihydroxyvitamin
D3 induced apoptosis that was detectable in the
adherent cells as well as by an increase in the proportion of apoptotic
cells floating in the medium. ALP activity has previously been used to
demonstrate that the induction of apoptosis in colonic tumor cells by
the differentiation agent, butyrate, is via differentiation (32
, 33)
. We used this same marker of colonic differentiation to
investigate whether 1,25-dihydroxyvitamin D3
also induced apoptosis via differentiation. ALP activity was increased
by 1,25-dihydroxyvitamin D3 treatment in both
the adherent- and floating-cell populations. The floating apoptotic
cells had higher levels of ALP activity than the adherent cells, which
indicated that apoptosis occurred by a process of differentiation and
apoptosis akin to terminal differentiation. The adenoma cell lines were
more sensitive to 1,25-dihydroxyvitamin
D3-induced apoptosis at the earlier time points,
but the carcinoma cell lines underwent greater apoptosis in response to
1,25-dihydroxyvitamin D3 after prolonged
treatment (5–7 days’ daily treatment with 1,25-dihydroxyvitamin
D3). In fact, apoptosis continued in the
carcinoma cell lines after 2 weeks of continuous daily treatment (data
not shown). The fact that colorectal carcinoma cells retain their
response to 1,25-dihydroxyvitamin D3 shows
promise for therapeutic strategies using noncalcemic analogues of the
hormone. Furthermore, the sensitivity of the adenoma cell lines to
1,25-dihydroxyvitamin D3-induced apoptosis may
be important in the prevention of colon cancer. All five of the cell
lines underwent apoptosis in response to 1,25-dihydroxyvitamin
D3, irrespective of p53 status (see "Materials
and Methods"), demonstrating p53-independent apoptosis.
Having shown that the active metabolite of vitamin
D3 induced apoptosis, we asked the important
question as to whether the analogue EB1089 inhibited growth by the same
mechanisms as the naturally occurring steroid. This analogue is
undergoing clinical trials for the treatment of a variety of cancers
because of its reduced hypercalcemic effects. EB1089 was investigated
under the same experimental conditions and using the same five cell
lines to determine whether it also induced apoptosis. The experiments
were designed to compare the response of the cells to EB1089 and
1,25-dihydroxyvitamin D3 and to investigate
whether EB1089 was growth inhibitory and/or induced apoptosis in
premalignant adenoma cells as well as in carcinoma cells. Studies with
EB1089 showed that this analogue is more effective than
1,25-dihydroxyvitamin D3 in inducing a
G1 arrest in colonic tumor cell lines. The
G1 arrest was evident at lower concentrations of
EB1089 compared with 1,25-dihydroxyvitamin D3
and also earlier during the treatment period. The adenoma cell lines
were markedly more sensitive to EB1089-induced cell cycle arrest than
the carcinomas. Furthermore, in the adenoma cell lines, RG/C2 and
AA/C1, and in the carcinoma cell line, PC/JW, EB1089 treatment produced
a greater reduction in attached cell yield at day 7 than
1,25-dihydroxyvitamin D3. The most striking
difference in the effect of the two agents was seen in the
PC/AA/C1 adenoma cell line, in which 10-10
M 1,25-dihydroxyvitamin
D3 induced only 9% reduction in cell
yield, whereas an equimolar concentration of EB1089 produced a 40%
reduction. In the AA/C1 and PC/JW cell lines, EB1089 was also more
effective in its induction of apoptosis than 1,25-dihydroxyvitamin
D3. Although the carcinoma cell line HT29 was
relatively resistant to apoptosis induced by EB1089 compared with
1,25-dihydroxyvitamin D3, EB1089 reduced the
adherent-cell yield of HT29 to a similar extent at concentrations of
10-10 to 10-7
M over the 7 days of treatment. The observation that EB1089
can induce apoptosis in adenoma as well as carcinoma cells supports the
idea of using EB1089 for colon cancer chemoprevention, particularly
given that, compared with 1,25-dihydroxyvitamin
D3, higher doses of EB1089 can be tolerated
in vivo.
To begin to determine how apoptosis is induced by
1,25-dihydroxyvitamin D3 and EB1089 in
colorectal adenoma and carcinoma cells, we selected four members of the
Bcl-2 family of apoptosis-regulatory proteins for study. The
proapoptotic protein Bak was consistently up-regulated in association
with apoptosis in all of the five colonic tumor cell lines. Bak
increases in response to EB1089 were often more pronounced and earlier
than in response to 1,25-dihydroxyvitamin D3.
That 1,25-dihydroxyvitamin D3 induces
differentiation and Bak-mediated apoptosis is consistent with the
gradient of expression of Bak in the crypt, with maximal expression in
the mature, differentiated cells at the top of the crypt
(44)
. In addition, we have data from SW620 cells treated
with 1,25-dihydroxyvitamin D3 and EB1089 for 7
days showing that the VDR protein levels were increased, consistent
with the binding of the ligand to the nuclear receptor (data not
shown). However, additional studies are required to determine whether
the induction of apoptosis by 1,25-dihydroxyvitamin
D3 is via the VDR nuclear receptor and whether
the VDR directly or indirectly induces Bak transcription.
EB1089 has been shown to down-regulate Bcl-2 in the breast cancer cell
line MCF7 (45)
. In the colonic tumor cell lines RG/C2 and
PC/JW, Bcl-2 was markedly down-regulated by EB1089; however, the
adenoma cell line AA/C1, which does not express Bcl-2, still underwent
apoptosis in response to both 1,25-dihydroxyvitamin
D3 and EB1089. Bcl-2 down-regulation is,
therefore, only partly responsible for the induction of apoptosis by
EB1089. Moreover, HT29 and SW620 did not show Bcl-2 down-regulation in
response to EB1089. On the contrary, there was a small increase in
Bcl-2 levels, which occurred late in the time course for HT29 and early
for SW620. Interestingly, this inversely correlated with the time
points at which most apoptosis occurred. Bcl-2 up-regulation may, in
part, confer some resistance to EB1089-induced apoptosis and counter
the effects of Bak up-regulation. It may represent a stress response by
the cells, as has been described previously (46)
.
In conclusion, both 1,25-dihydroxyvitamin D3
and EB1089 induce apoptosis in colorectal tumor cells. Apoptosis seems
to occur subsequent to the induction of enterocytic (absorptive cell)
differentiation. Up-regulation of Bak in response to
1,25-dihydroxyvitamin D3 and EB1089 in five
different colorectal tumor cell lines occurs in association with
apoptosis, which suggests that this protein mediates at least part of
the apoptotic response to these agents and that apoptosis is induced by
similar mechanisms for both 1,25-dihydroxyvitamin
D3 and its analogue, EB1089. Interest has focused
on EB1089 because of its reduced hypercalcemic effects, which mean
that, compared with 1,25-dihydroxyvitamin D3,
higher doses of EB1089 can be achieved in vivo. In addition
to inducing apoptosis, the analogue EB1089 induces a more pronounced
growth arrest in the G1 phase of the cell cycle
than 1,25-dihydroxyvitamin D3, particularly in
the premalignant adenoma cells. These studies suggest that EB1089 may
be an effective chemopreventive agent for individuals at high risk of
developing colorectal cancer. Furthermore, EB1089 induces apoptosis
(the extent of which increases with time of treatment) in colorectal
cancer cells, including cells with mutant p53 and the SW620 cell line,
which was derived from a lymph node metastasis (28)
.
Therefore, EB1089 shows potential for the treatment of colorectal
cancer in conjunction with current chemotherapy. Vitamin D analogues
such as EB1089 have potential as therapeutic agents for colorectal
cancer, and a greater understanding of their mechanisms of action may
enable more effective clinical application.
|
FOOTNOTES |
|---|
1 To whom requests for reprints should be
addressed, at Cancer Research Campaign, Colorectal Tumour Biology
Research Group, Department of Pathology and Microbiology, School of
Medical Sciences, University of Bristol, University Walk, Bristol BS8
1TD, United Kingdom. 
2 The abbreviations used are: VDR, vitamin D
receptor; VDRE, vitamin D responsive element; ALP, alkaline
phosphatase.
Received 10/ 1/99. Accepted 3/ 3/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSMeasurement of Differentiation:...RESULTSDISCUSSIONREFERENCES |
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U. Maurer, F. Jehan, C. Englert, G. Hubinger, E. Weidmann, H. F. DeLuca, and L. Bergmann The Wilms' Tumor Gene Product (WT1) Modulates the Response to 1,25-Dihydroxyvitamin D3 by Induction of the Vitamin D Receptor J. Biol. Chem., February 2, 2001; 276(6): 3727 - 3732. [Abstract] [Full Text] [PDF]
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T. F. McGuire, D. L. Trump, and C. S. Johnson Vitamin D3-induced Apoptosis of Murine Squamous Cell Carcinoma Cells. SELECTIVE INDUCTION OF CASPASE-DEPENDENT MEK CLEAVAGE AND UP-REGULATION OF MEKK-1 J. Biol. Chem., July 6, 2001; 276(28): 26365 - 26373. [Abstract] [Full Text] [PDF]
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