Yonago Acta medica 1999;42:113–124

Ethanol Induces Apoptosis in Human Gastric Carcinoma Cells: The
Role of Apoptosis-Related Molecules
Noriko Kasagi
First Department of Pathology, Faculty of Medicine, Tottori University, Yonago 683-0826,
Japan
Ethanol triggers apoptosis in a variety of mammalian cultured cells. Here, ethanolinduced apoptosis in five human gastric carcinoma cell lines was examined. Trypan
blue-positive cells were detected in more than 77% of 10% ethanol treated cells at 6 h
in all the cell lines. The apoptosis, which had a typical morphology and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) signals
were induced with 3% and 5% ethanol treatments. The apoptotic cells were detected
in 36% of MKN-74, 54% of MKN-45 which have wild-type p53, and 37% of TMK-1
cells at 48 h with 5% ethanol, in contrast to less than 18% of MKN-28 and KATO-III
cells. The DNA ladder appeared in the MKN-74, MKN-45 and TMK-1, but not in the
MKN-28 and KATO-III. The expression of apoptosis-associated molecules p53, Bcl-2,
Bax and c-Myc proteins was analyzed in the ethanol-treated cells. The levels of p53,
Bcl-2 and Bax proteins were not changed in the ethanol-induced apoptosis. MKN-28
and KATO-III showed higher expressions of Bcl-2 protein compared to the other celllines, and the down-regulation of c-Myc protein after 1.5 h of ethanol treatment. Calcium
antagonists verapamil or nifedipine decreased apoptotic cells in ethanol treated MKN74, MKN-45 and TMK-1. These results suggest: i) 3% and 5% ethanol induced apoptosis
via the influx of extracellular calcium, but were not changed the expression levels of
p53 and c-Myc protein, while 10% ethanol provoked necrosis in these cell lines, and ii)
the apoptosis resistance by ethanol might be implicated in the higher expression of Bcl2 protein and down-regulation of c-Myc protein as shown in MKN-28 and KATO-III.
Key words: apoptosis; gastric carcinoma cell line; ethanol; calcium; c-Myc protein

Apoptosis is a form of cell suicide and plays amajor role in embryogenesis, the regulation of
the immune system, and carcinogenesis (Kerr etal., 1972, 1994; Wyllie, 1980, 1981; Ucker, 1991;
Williams, 1991; Anilkumar et al., 1992; Cohen,
1993; Hockenbery, 1995). The morphologicalfeatures of apoptosis are cell shrinkage, loss ofnormal contacts and condensation of the nuclear chromatin. The apoptotic cells with nuclear fragmentation subsequently break down
into multiple apoptotic bodies, which are phagocytosed rapidly by adjacent cells or macrophages (Kerr et al., 1972; Wyllie et al., 1984).
Biochemically, deoxyribonucleic acid (DNA)isolated from apoptotic cells shows a characteristic “ladder” structure on agarose gel electrophoresis (Wyllie, 1980).
Ethanol has been shown to induce the apoptosis of thymocytes, lymphocytes, hepatocytes,
and tumor cell lines in vitro ( Lennon et al.,
1991; Ewald and Shao, 1993; Slukvin andJerrells, 1995; Kravtsov and Fabian, 1996;
Kikuchi et al., 1997; Kurose et al., 1997). Ethanol affects a signal transduction pathway inthymocytes by altering the cellular calcium
level and increasing protein kinase C activity(Shao et al., 1995; Slukvin and Jerrells, 1995).
However, the signal transduction pathways

Abbreviations: DAB, diaminobenzidine; DMSO, dimethyl sulfoxide; dUTP, deoxyuridine 5'-diphosphate; 5FU, 5-fluorouracil; PBS, phosphate-buffered saline; TdT, terminal deoxynucleotidyl transferase; TUNEL,
TdT-mediated dUTP-biotin nick end-labeling113

N. Kasagi

underlying ethanol-induced apoptosis still remains obscure. It has also been shown thatethanol causes rat or rabbit gastric mucosal cell
damage (Oates and Hakkinen, 1988; Tepperman
et al., 1991) and apoptosis (Piotrowski et al.,1997). Tarnawski and colleagues (1990) havesuggested that both ethanol penetration andinflux of extracellular calcium are necessary for
isolated human gastric gland cell injury byethanol. Livraghi and colleagues (1991) havereported that percutaneous ethanol injection
was performed for the treatment of metastatic
liver cancer, including gastric carcinoma, andshowed reduction in tumor size. It has been
also reported that apoptosis occurred in humangastric carcinomas in both p53 gene-dependentand -independent manners (Kasagi et al., 1994;
Ishida et al., 1997; Ikeda et al., 1998). However, it has not been known whether ethanol induces apoptotic cell death in human gastriccarcinoma cells. In addition, the roles of apoptosis-related genes in the process of ethanolinduced apoptosis are as yet poorly understood.
In the present study, the induction of apoptosis
with ethanol in gastric carcinoma cell lines wasstudied. The apoptosis-related molecules wereanalyzed to define the mechanism andregulation of the ethanol-induced apoptosis.

Materials and Methods

Cell lines
MKN-74 and MKN-28 (Motoyama et al., 1986)
were each derived from a well-differentiatedadenocarcinoma, MKN-45 (Motoyama et al.,1986) and TMK-1 (Ochiai et al., 1985) frompoorly differentiated adenocarcinomas, and
KATO-III (Sekiguchi et al., 1978) from a signet
ring cell carcinoma. MKN-74 and MKN-45
have wild-type p53 genes, while MKN-28 andTMK-1 carry mutated p53 genes (Matter et al.,1992). The p53 gene of KATO-III is completely deleted (Matter et al., 1992). All cell lines
were cultured in RPMI 1640 medium (Cosmo
Bio, Tokyo, Japan), supplemented with 200
mmol/L glutamine, 100 U/mL penicillin and
10% fetal bovine serum (Bio Whittaker,

Walkersville, MD) at 37˚C in a humidified 5%
CO2 atmosphere.

Induction of cell death
The cells were plated to each experiment at adensity of 2 × 105 cells/mL. After 24 h of culture, the medium was replaced with fresh medium containing various concentrations (1%, 3%,
5% and 10%) of ethanol. The cells were thenincubated at 37˚C in a humidified 5% CO 2
atmosphere. The cells without ethanol wereserved as controls. All experiments were performed on cells in exponential phase of growth.

Trypan blue staining
The cells were plated at 5 × 104 cells/mL. For acount of the number of dead cells, the trypan
blue-positive cells were measured in 3 different
cultures with the trypan blue exclusion test, andthe mean value of percentage of positive cells
was calculated.

Papanicolaou stain
For morphological examinations, cells wereprepared with Auto Smear CF-120 (Sakura,Tokyo) and evaluated with Papanicolaoustaining. Apoptotic and necrotic cells wereidentified using the criteria described by Kerrand colleagues (1972), Wyllie and colleagues(1980) and Searle and colleagues (1982).

TdT-mediated dUTP-biotin nick endlabeling (TUNEL) method
DNA breaks in cells were detected with a modified TUNEL method (Kasagi et al., 1996). The
cells were scraped using a cell scraper (Falcon,
Lincoln Park, NJ) and suspended in 100%
methanol at 4˚C for 30 min and then dropped
onto silane-coated glass slides (Muto Pure
Chemicals, Tokyo). Preparations were treatedwith 1% H2O2 solution for 20 min. The cells on
glass slides were covered with TdT buffer solution (100 mmol/L potassium cacodylate, 2mmol/L cobalt chloride, 0.2 mmol/L dithiothreitol, pH 7.2) containing 0.3 U/µL TdT

114

Ethanol induces apoptosis in carcinoma

A

MKN-74

(%)

B

80

60

40

20

MKN-45(%)
100

Trypan blue-positive cells

Trypan blue-positive cells

100

80

60

40

20

0

0
0 3 6

12

24

48

(h)

0 3 6

Incubation time

C

D

(%)

48

(h)

48

(h)

(%)

Trypan blue-positive cells

Trypan blue-positive cells

TMK-1

60

40

20

0

100

80

MKN-28

60

40

20

0
0 3 6

12

24

48

(h)

0 3 6

12

24

Incubation time

Incubation timeE

24

Incubation time

100

80

12

(%)

Trypan blue-positive cells

100

80

KATO-III

60

Control
1% EtOH
3% EtOH
5% EtOH
10% EtOH

40

20

0
0 3 6

12

24

48

(h)

Incubation time
Fig. 1. Induction of cell death in the human gastric carcinoma cell lines MKN-74 (A), MKN-45 (B), TMK-1(C), MKN-28 (D) and KATO-III (E) treated with ethanol. Each cell line was exposed to 0% (control), 1%,
3%, 5% and 10% ethanol and cultured for the indicated time. Results are expressed as percentages of trypan
blue-positive cells, as determined by the cells’ ability to exclude trypan blue dye. Data are the means of three
separate experiments.

115

N. Kasagi(Gibco BRL Life Technologies, Gaithersburg,MD) and 0.04 nmol/µL biotinylated dUTP
(Boehringer Mannheim, Mannheim, Germany)and incubated in a humidified atmosphere at
37˚C for 90 min. Cells were then washed with
TB buffer (300 mmol/L sodium chloride, 30
mmol/L sodium citrate) and then with phosphatebuffered saline (PBS). After incubation withperoxidase-labeled streptavidin for 30 min, thecells were developed in a mixture of 0.02%
diaminobenzidine (DAB) and 0.02% H 2O 2
solution, and counterstained with Alcian green.
As the negative control, cells on glass slides
were treated with only TdT buffer solution.
Cells were considered positive for TUNEL
when their nuclei were labeled on high-power
fields except for faint signals. The proportionof TUNEL-positive cells was calculated in atleast 1,000 cells and expressed as a percentage.
The results were expressed as the mean and SD
in 3 separate experiments.

DNA isolation and electrophoresis
Cells were collected and suspended at a densityof 2 × 106 cells/mL in lysis buffer (10 mmol/L
Tris-HCl buffer, pH 8.0, 5 mmol/L EDTA, 100
mmol/L sodium chloride) containing 10 µg/mL
proteinase K (Wako Pure Chemicals, Osaka,
Japan) and 0.2% sodium dodecyl sulfate andincubated at 37˚C overnight. The crude DNA
preparations were extracted with phenol:chloroform:isoamyl alcohol (25:24:1), and then withchloroform:isoamyl alcohol (24:1). RNase A(Sigma, St. Louis, MO) was then added to aconcentration of 100 µg/mL followed byincubation at 37˚C for 2 h. The DNA preparations were extracted again and precipitated inethanol at –80˚C. The DNA precipitates were
resuspended in TE buffer (10 mmol/L Tris-HCl
buffer, pH 7.4, 1 mmol/L EDTA).
DNA samples of 1 µg were then loaded onto
a 2% agarose gel, and electrophoresis was
carried out for 1.5 h at 50 V. As the size marker,
123 base pair DNA Ladder (Gibco BRL Life
Technologies) was used. DNA was stainedwith 1 µg/mL ethidium bromide and visualized
by fluorescence in UV light.

Western blot analysis
Cells were solubilized in lysis buffer (50 mmol/
L Tris-HCl buffer pH 7.4, 125 mmol/L NaCl,
0.1% NP-40, 5 mmol/L NaF, 1 mmol/L PMSF,
1 ng/mL leupeptin, 10 ng/mL soybean trypsin
inhibitor, 1 ng/mL aprotinin, 10 ng/mL N-tosylL -phenylalanyl chloromethyl ketone) for 60
min on ice. The protein concentration was determined by means of the Bradford protein
assay (Bio-Rad Laboratories, Richmond, CA)with bovine serum albumin as the standard.
Samples (50 µg) were resolved by electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel and electroblotted onto a polyvinylidene difluoride filter (Millipore, Bedford,
MA). The filters were incubated with the primary antibodies at 4˚C overnight and then withperoxidase-labeled anti-mouse IgG (diluted
1:1,000; Medical and Biological Laboratories,
Nagoya, Japan) or anti-rabbit IgG antibody(diluted 1:2,000; Medical and Biological Laboratories) in the secondary reaction. The primary
antibodies used were anti-p53 (diluted 1:100;
1801; MEDAC, Hamburg, Germany), anti-Bcl2 (diluted 1:100; 124; Dako, Copenhagen,Denmark), anti-c-Myc (diluted 1:50; Ab-1;
Oncogene Research Products, Cambridge, MA)
monoclonal antibodies and anti-Bax (diluted
1:1,000; P-19; Santa Cruz Biotechnology, Santa
Cruz, CA) polyclonal antibody. The immune
complex was detected using an enhanced
chemiluminescence system (Amersham,
Buckinghamshire, United Kingdom).

Effects of calcium antagonists on ethanol-induced apoptosis
After being seeded in 60 mm tissue culture
dishes and cultured for 24 h, cells were treatedwith verapamil (Sigma) or nifedipine (Sigma)
dissolved in dimethyl sulfoxide (DMSO) for 30
min, and then ethanol (3% or 5%) was added.
After 12 h or 24 h of culture respectively, thepercentage of TUNEL-positive cells was calculated as described above.
For the identification of the optimum concentrations of verapamil and nifedipine, pre-116

Ethanol induces apoptosis in carcinoma

A

B

(%)

MKN-74

(%)

80

60

40

20

MKN-45100

TUNEL-positive cells

TUNEL-positive cells

100

80

60

40

20

0

0
0 3 6

12

24

48

0 3 6

(h)

D

(%)

TMK-1

80

60

40

20

0

(h)

48

(h)

MKN-28

80

60

40

20

0
0 3 6

12

24

48

(h)

0 3 6

Incubation time

E

48

(%)
100

TUNEL-positive cells

TUNEL-positive cells

100

24

Incubation time

Incubation time

C

12

12

24

Incubation time(%)

TUNEL-positive cells

100

KATO-III
80

60

40

1% EtOH
3% EtOH
5% EtOH
10% EtOH

20

0
0 3 6

12

24

48 (h)

Incubation time

Fig. 2. Time course of TUNEL-positive cells in MKN-74 (A), MKN-45 (B), TMK-1 (C), MKN-28 (D) and
KATO-III (E) treated with ethanol. Each cell line was exposed to 0% (control), 1%, 3%, 5% and 10%
ethanol. Results are expressed as percentages of TUNEL-positive cells, as stained by the TUNEL method.
Data are the means of three separate experiments.

117

N. Kasagi

liminary investigations were carried out whichgave minimum trypan blue- and TUNEL-positive cells with or without ethanol. Verapamil or
nifedipine was added at the final concentrationof 100 µmol/L (TMK-1 and MKN-74) or 200µmol/L (MKN-45, MKN-28 and KATO-III).
Control cultures contained 0.2% DMSO inplace of calcium antagonists.
The significance of differences between
means was estimated using Student’s t-test.

Results

Induction of cell death by ethanol treatment
The effects of ethanol on cell death were determined in the gastric carcinoma cell lines at various timepoints of treatment and ethanol concentrations (Fig. 1). The percentages of trypan

blue-positive cells increased in a dose-dependent manner in the cell lines. With 10% ethanol
treatment, the proportion of trypan blue-positive cells rapidly increased to more than 77% at
6 h of culture in all the cell lines. Treatmentswith 3% and 5% ethanol induced cell death in atime-dependent manner in all cell lines except
MKN-28. No effect was observed with 1%
ethanol.
Figure 2 shows the effects of ethanol on the
induction of DNA fragmentation, which wasestimated by the TUNEL method. In the celllines MKN-74, MKN-45 and TMK-1, the percentages of TUNEL-positive cells increased ina time-dependent manner when the cells weretreated with 5% ethanol. The signals of MKN74, MKN-45 and TMK-1 were in 35.7 ± 13.8%,
54.2 ± 15.2% and 37.3 ± 13.6% at 48 h of culture, respectively. However, the percentages of
TUNEL-positive cells in TMK-1 treated with
3% ethanol were 32.3 ± 10.1% at 12 h and 15.6

Fig. 3. Morphological features of apoptosis induced by ethanol in MKN-74 (A, B) and TMK-1 (C, D) cells
stained with Papanicolaou stain (A, C) and the TUNEL method (B, D). MKN-74 cells were exposed to 5%
ethanol for 48 h (A, B) and TMK-1 cells were exposed to 3% ethanol for 12 h (C, D). A, C: Cells had nuclear
condensation, fragmentation or apoptotic bodies. B, D: TUNEL signals were found in the fragmented andcondensed nuclei. A cell with a condensed nucleus had no TUNEL signals (arrow). Scale bar = 10 µm.

118Ethanol induces apoptosis in carcinoma

B

M
ar
C ker
on
3% tro
l
2
5% 4
h
10 24
% h
3% 24
h
5% 48
h
10 48
% h
48
h

MKN-74stained materials. TUNEL signals with various
intensity were found in the nuclei (Fig. 4).

MKN-45
M
ar
C ker
on
3% tro
l
2
5% 4
h
10 24
% h
3% 24
h
5% 48
h
4
10 8
% h
48
h

A

Fig. 4. Necrotic cells stained by the TUNEL method. TMK-1 cells were exposed to 10% ethanol for
48 h and stained by the TUNEL method. They hadvarious intensities of TUNEL signals. Scale bar =10
µm.

C

(bp)

(bp)

(bp)

1230–

1230–

1230–

615–

615–

615–

123–

123–

123–

E

(bp)

(bp)

1230–

1230–

615–

615–

123–

123–

KATO-III

M
ar
C ker
on
3% tro
l
2
5% 4
h
10 24
% h
3% 24
h
5% 48
h
4
10 8
% h
48
h

MKN-28

M
ar
C ker
on
3% tro
l
2
5% 4
h
10 24
% h
3% 24
h
5% 48
h
10 48
% h
48
h

D

TMK-1
M
ar
C ker
on
3% tro
l
2
5% 4
h
10 24
% h
3% 24
h
5% 48
h
4
10 8
% h
48
h

± 3.7% at 48 h of culture. On the other hand, the
TUNEL signals were 4.7 ± 0.8% of MKN-28
and 17.2 ± 7.6% of KATO-III cells at 48 h ofculture with 5% ethanol. The percentages of
TUNEL-positive cells also increased with 10%
ethanol in a time-dependent manner in all celllines except MKN-74.
Morphologically, the typical features of
apoptosis were seen in the cells treated with 3% or
5% ethanol with Papanicolaou stain (Figs. 3A and
C). TUNEL signals were found in condensed or
fragmented nuclei, and also in some normallooking nuclei (Figs. 3B and D). A few cellswith fragmented or condensed nuclei had no
TUNEL signals in the TMK-1 (Fig. 3D) and
KATO-III (data not shown) cultures. Cells cultured with 10% ethanol exhibited a necrotic
appearance in all of the cell lines. These cells
had nuclei that were reduced in size and indistinct vacuolated cytoplasm on Papanicolaou-Fig. 5. DNA fragmentation analysis by agarose-gel electrophoresis. DNA fragmentation in MKN-74 (A),MKN-45 (B), TMK-1 (C), MKN-28 (D) and KATO-III (E) cells treated with ethanol. Each cell line was
exposed to 0% (control), 3%, 5%, and 10% ethanol for indicated time. The DNA ladder of apoptosis wasseen in MKN-74 (A: 5% ethanol 24 h and 48 h), MKN-45 (B: 5% ethanol 24 h and 48 h) and TMK-1 cells (C:3% ethanol 12 h and 24 h, 5% ethanol 24 h). The DNA smear pattern of necrosis was seen in cell lines
exposed to 10% ethanol. Marker, 123 base pair DNA ladder molecular size marker.

119

N. Kasagi

MKN-74

MKN-45

TMK-1

C

9

12

15

18

21

24

36

h

C

9

12

15

18

21

24

36

h

C

3

6

9

12

15

24

h

C

9

12

15

18

21

24

36

h

C

9

12

15

18

21

24

36

h

C

3

6

9

12

15

24

h

← 53 kDa

p53← 26 KDa

Bcl-2
C

9

12

15

18

21

24

36

h

C

9

12

15

18

21

24

36

h

C

3

6

9

12

15

24

h

C

9

12

15

18

21

24

36

h

C

9

12

15

18

21

24

36

h

C

3

6

9

12

15

24

h

← 21 kDa

Bax← 67 kDa

c-Myc

KATO-III
C

6

12

24

36

48

MKN-28
h

C

6

12

24

36

48

h

← 53 kDa

p53
C

6

12

24

36

48

h

C

6

12

24

36

48

h

C

6

12

24

36

48

h

C

6

12

24

36

48

h

← 26 KDa

Bcl-2← 21 kDa

Bax
C

0.5

1

1.5

2

3

6

12

24

h

C

0.5

1

1.5

2

3

6

12

24

h

← 67 kDa

c-Myc

Fig. 6. Western blot analysis of p53, Bcl-2, Bax and c-Myc proteins in MKN-74, MKN-45, TMK-1, KATOIII and MKN-28 cells. MKN-74, MKN-45, KATO-III and MKN-28 cells were incubated with 5% ethanol
and TMK-1 cells were incubated with 3% ethanol for indicated times. C, without ethanol (control).

Analysis of DNA fragmentation by agarose gel electorophoresis
To determine whether the DNA degradation
was apoptosis or necrosis, the DNAs obtained
from each cell line were analyzed by agarose
gel electrophoresis. The “ladder” pattern, ahallmark of apoptosis, clearly appeared on theelectrophoresis of MKN-74 and MKN-45 cells
treated with 5% ethanol and TMK-1 cells treated with 3% ethanol (Figs. 5A, B and C). No
ladder pattern was observed in all DNAs from
MKN-28 and KATO-III cells (Figs. 5D and E).
DNA from all five of the cell lines treated with
10% ethanol showed a smear-like, non-specificdegradation, regarded as characteristic of
necrosis (Fig. 5).

Expression of p53, Bcl-2, Bax and c-Myc
protein (Fig. 6)
For the determination of the expression of theapoptosis-related genes, cells were cultured
with 3% or 5% ethanol, and then cellular lysates

were analyzed by Western blot. The expression
levels of p53, Bcl-2 and Bax proteins did notchange during the ethanol exposure in any of
the cell lines. The expression of p53 protein
was higher in the TMK-1 and MKN-28 cells,
which carry a mutated p53 gene, compared to
the MKN-45 and MKN-74 cells, which carry
the wild-type p53 gene. The KATO-III cells(with the p53 gene deleted) did not express p53
protein. The expression level of Bcl-2 protein
was higher in the MKN-28 and KATO-III cells
than in the other three lines, in the presence or absence of ethanol. MKN-74, MKN-45 and TMK-1
expressed c-Myc protein in the presence or absence of ethanol. The c-Myc protein expressions in MKN-28 and KATO-III were apparently down-regulated after 1.5 h with 5% ethanol
treatment and only slightly detected at 24 h.

Effect of calcium antagonists on ethanol-induced apoptosis (Fig. 7)
The role of calcium on the induction of apoptosis
was examined using the calcium ion channel

120

Ethanol induces apoptosis in carcinoma

TUNEL-positive cells

50

(%)

40

30

20

*
*
*

**

10

0

Ethanol(%)
Verapamil (µmol/L)
Nifedipine (µmol/L)

–

5

5

–

5

5

–

3

3

–

5

5

–

5

–

– 100 –

5–

– 200 ––

– 100 ––

– 200 ––

– 200 –

–

–

–

–

–

–

–

–

–

–

– 100

MKN-74

5– 200

MKN-45

3

– 100

TMK-1

5

– 200

MKN-28

5

5

– 200

KATO-III

Fig. 7. Effect of calcium ion channel antagonists, verapamil and nifedipine, on apoptosis induced by ethanol.
TMK-1 cells were treated with ethanol with/without a calcium ion channel antagonist for 12 h, and the othercell lines were treated for 24 h. Results were shown as the percentage of TUNEL-positive cells. The data aremean ± SD of three separate experiments. *P < 0.05, **P < 0.01 compared with ethanol only-treated cells.

antagonists verapamil and nifedipine (Tepperman
and Whittle, 1991). In the MKN-74 cells, incubation with verapamil or nifedipine significantly reduced the percentages of ethanolinduced TUNEL-positive cells. MKN-45 andTMK-1 cells pretreated with verapamil or
nifedipine showed decreased TUNEL-positive
cells in the ethanol treated cells. The antagonists did not reduce the percentages of TUNELpositive cells in the MKN-28 and KATO-III
cell lines.

Discussion
In this study, it was demonstrated that ethanol
induced cell death via apoptosis or necrosis in
human gastric carcinoma cell lines in a concentration-dependent manner. The characteristic
morphology of apoptosis and TUNEL signals
were found in all five cell lines treated with 3%
or 5% ethanol, while DNA ladder formation,
one of the features of apoptosis (Wyllie, 1980),
was confirmed in MKN-74, MKN-45 and TMK1, but not in MKN-28 and KATO-III cells treatedwith 5% or 3% ethanol. More than 77% of thecells showed necrotic features with DNA smear
patterns in all of the cell lines treated with 10%
ethanol at 6 h after the treatment. Trypan bluepositive cells were found in more than 91%, and
TUNEL-positive cells were detected in morethan 21% of all cell lines treated with 10% ethanol, at 48 h of culture. These results indicatedthat TUNEL signals are also observed in necrotic cells, as reported by Kressel and Groscurth(1994). The present results could be roughlysummarized that 3% or 5% ethanol induces
apoptosis, whereas 10% ethanol causes necrotic
cell death in gastric carcinoma cells. The results are consistent with the previous reports
which described apoptosis induced by ethanol
in HL-60 cells, thymocytes and lymphocytes(Lennon et al., 1991; Slukvin and Jerrells, 1995;
Kravtsov and Fabian, 1996).
A variety of protein molecules or oncogenes
and suppressor genes are involved in the process of apoptosis. p53 gene product has beenshown to be required for the induction of theapoptotic pathway triggered by oncogeneous
activation and cytotoxic genes (Lowe et al.,1993a, 1993b). The product may sensitize
damaged cells to apoptosis, acting to prevent121

N. Kasagi

the propagation of transforming mutations.
Fritsche and colleagues (1993) demonstrated
that anticancer agents such as cisplatine, mitomycin C, etoposide and 5-fluorouracil (5-FU),
as well as energy-rich radiation act on cellularDNA. These agents induced a nuclear accumulation of p53 protein followed by apoptosis.
Osaki and colleagues (1997) have reported that
5-FU induced apoptosis of the human gastriccarcinoma cell lines MKN-45 and MKN-74(the wild-type p53 gene), but not in MKN-28(the mutated p53 gene) and KATO-III (lacking
the p53 gene). The induction of p53 protein expression was noted in the former two, but not in
the latter two cell lines. In the present study,
p53 protein expression levels in all five of thecell lines were unchanged in the process of
ethanol-induced apoptosis. Therefore, the results have indicated that p53 protein does notplay the role in ethanol-induced apoptosis.
Ethanol also did not alter the expression
levels of Bcl-2 and Bax proteins in the processof apoptosis induction in any of the cell lines.
What’s interesting is that 3% or 5% ethanol
hardly induced apoptosis in MKN-28 and KATOIII cells, which showed higher expression levelsof Bcl-2 protein compared to the other three celllines. Our results indicate that higher expression of Bcl-2 protein in these gastric carcinoma
cells may be associated with the inhibition of
ethanol-induced apoptosis. A higher expression of Bcl-2 protein inhibited the induction of
apoptosis with various agents in a variety of
myeloid leukemia cells (Campos et al., 1993;
Lotem and Sachs, 1993).
Overexpression of the c-myc gene has beenshown to be strongly associated with various
human cancers. The constitutive activation ofc-Myc protein of cultured cells leads to proliferation and cellular transformation. Moreover,overexpression of c-Myc protein might accelerate cell death via apoptosis (Askew et al.,
1991; Evan et al., 1992) in a p53-dependent or-independent mechanism in epithelial cells(Sakamuro et al., 1995). In the present study,
the expression level of c-Myc protein did notchange throughout the course of apoptosis in
MKN-74, MKN-45 and TMK-1 cells treatedwith 5% ethanol. Thus, the results do not indi-cate that c-Myc protein plays an important rolein the apoptosis signal pathway in these celllines. On the other hand, the c-Myc protein expression was reduced at 1.5 h and the reduction
was sustained up to 24 h in the two apoptosisresistant cell lines MKN-28 and KATO-III.
Dong and colleagues (1997) reported that thelower expression of c-Myc protein is strongly
associated with the resistance to etoposideinduced apoptosis in human monocytic leukemia cells, and that c-Myc protein plays a role incellular susceptibility to death receptor-mediated and chemotherapy-induced apoptosis. It
may be that the reducing c-Myc protein expression is also related to the resistance to ethanolinduced apoptosis.
Tarnawski and colleagues (1990) have reported that the influx of extracellular calcium isnecessary for human gastric gland cell injury.
Alterations in intracellular calcium ion homeostasis are commonly observed during apoptosis,
and insights into how calcium ions might regulate the caspases and other components of thepathway are now emerging (McConkey andOrrenius, 1997). The presence of extracellular
calcium was found to be necessary for the
induction of apoptosis with ethanol (Lennon etal., 1991; Slukvin and Jerrells, 1995), and thecytosolic free calcium increase triggered byethanol may be a part of the signal pathways
leading to apoptosis (Shao et al., 1995). In thepresent study, verapamil and nifedipine, which
inhibit calcium entry into the cell, partiallyinhibited the induction of apoptosis by ethanol
in MKN-74, MKN-45 and TMK-1 cells, whichhave a lower expression of Bcl-2 protein. It isreasonable to assume that a calcium influx
through calcium ion channels by ethanol triggers apoptosis, and that cytosolic free calcium
in apoptotic cells increases and regulates several steps in the apoptotic pathway. In addition,
the calcium antagonists did not inhibit theinduction of apoptosis by ethanol in MKN-28
and KATO-III cells, which have a higher Bcl-2
protein expression. Lam and colleagues (1994)
have shown that an overexpression of Bcl-2
protein interferes with thapsigargin-induced
calcium ion mobilization from the endoplasmic
reticulum in the mouse lymphoma W.Hb12

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Ethanol induces apoptosis in carcinoma

cells. Additionally, Marin and colleagues(1996) reported that Bcl-2 protein blocks theaccumulation of calcium ion in the nucleus incells exposed to tumor necrosis factor orthapsigargin. Therefore, the overexpression of
Bcl-2 protein in MKN-28 and KATO-III cells
might interfere with an ethanol-induced calcium ion mobilization or the accumulation of
calcium ion in the nucleus.
In summary, this initial study indicated thatless than 5% ethanol induced apoptosis by the
influx of calcium ions across the plasma membrane and that p53 and c-Myc protein were notassociated with the process of apoptosis, while
10% ethanol provoked necrosis in five humangastric carcinoma cell lines. The two ethanolinduced apoptosis-resistant cell lines showed ahigher expression of Bcl-2 protein, the downregulation of c-Myc protein, and no effects ofcalcium ion channel antagonists. The preciserole of these molecules and intracellular signaltransduction in the process of ethanol-induced
apoptosis await further investigation, for the
clarification of the mechanism and regulation of
apoptosis in human gastric carcinomas.
Acknowledgments: I wish to express my sincere
gratitude to Prof. Hisao Ito, First Dept. of Pathology,
Faculty of Medicine, Tottori University, for his kindadvice and reading of the manuscript. I would like tothank Prof. Tadashi Terada, Second Dept. of Pathology, Faculty of Medicine, Tottori University andProf. Takeshi Sairenji, Dept. of Biosignaling, Schoolof Life Science, Faculty of Medicine, Tottori University, for valuable suggestions. I also thank Mr.
Norihisa Itaki for his technical advice.
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(Received December 21, 1998, Accepted January 14, 1999)

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