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Zhong-Ying
Shen, Ming-Hua Chen, Jian Shen, Wei-Jie Cai, Department of
Pathology
Wen-Ying Shen, Department of Chemistry Medical College of
Shantou University, Shantou, 515031, Guangdong, China
Zeng Yi, Institute of Virology, Chinese Academy of Preventive
Medicine. Beijing, 100052, China
Supported by the National Natural Science Foundation of
China. No. 39830380
Correspondence to: Dr. Zhong-Ying Shen, Department of
Pathology, Medical College of Shantou University, 22 Xinling Road.
Shantou 515031, Guandong Province, China. Zhongyingshen@yahoo.com.
Telephone:
+86-754-8538621 Fax: +86-754-8537516
Received 2001-08-09 Accepted 2001-11-12
Abstract
AIM: To
Quantitatively analyze the nitri oxide (NO) and Ca2+ in
apoptosis of esophageal carcinoma cells induced by arsenic trioxide
(As2O3).
METHODS:
The cell line SHEEC1, a malignant esophageal epithelial cell induced
by HPV in synergy with TPA in our laboratory, was cultured in a
serum-free medium and treated with As2O3.
Before and after administration of As2O3, NO
production in cultured medium was detected quantitatively using the
Griess Colorimetric method. Intracellular Ca2+ was
labeled by using the fluorescent dye Fluo3-AM and detected under
confocal laser scanning microscope (CLSM), which was able to acquire
data in real-time enabling Ca2+ dynamics of individual
cells in vitro . The apoptotic cells were examined under
electron microscopy.
RESULTS:
Intracellular concentration of Ca2+ increased from 1.00
units to 1.09-1.38 units of fluorescent intensity at As2O3
treatment and NO products subsequently released from As2O3-treated
cells increased from 0.98-1.00 ×10-2μmol·L-1
up to 1.48-1.52×10-2μmol·L-1 and
maintained in a high level continuously. Finally apoptosis of cells
occurred,chromatin being agglutinated, cells shrunk, nuclei became
round and mitochondria swelled.
CONCLUSION:
Ca2+ and NO increased with cell damage and apoptosis in
cells treated by As2O3. The Ca2+ is
an initial messenger to the apoptotic pathway. To investigate Ca2+
and NO will be a new direction for studying the apoptotic signaling
messenger of the esophageal carcinoma cells induced by As2O3.
Shen
ZY, Shen WY, Chen MH, Shen J, Cai WJ, Yi Z.Nitric oxide and calcium
ions in apoptotic esophageal carcinoma cells induced by arsenite.
World J Gastroenterol 2002;8(1):40-43
INTRODUCTION
Arsenic
trioxide (As2O3) has been proved to be a
genotoxic and a carcinogenic agent[1-6]. Previous studies
also showed that As2O3 induced cellular
apoptosis in leukemia[7-15], in cancer cells of head and
neck[16] and other cancer cells[17-22]. So As2O3
has antitumoral effect. We found that As2O3
induced apoptosis in esophageal squamous carcinoma cells[23].
The pathomorphological changes induced by As2O3
revealed that cells became smaller and shrank, nucleus rounded up,
chromatin agglutinated and marginated, the nuclear membrane broke
down followed by degenerative changes and cell mortality. All these
changes indicated typical morphological changes of apoptosis[24,
25]. Mitochondria, an important cellular apparatus, is related
to cell breathing, oxygen metabolism, enzyme activity and energy
supply. Our data demonstrated that the primary target of As2O3
inducing apoptosis of esophageal carcinoma cells might be the
mitochondria[26]. It is likely that As2O3
is a mitochondriotoxic agent[27, 28]. At the early stage
of cellular apoptosis induced by As2O3, the
mitochondria generated morphological and functional changes[29,
30].
NO
exerts a wide range of its biological properties via its interaction
with mitochondria and NO mediated mitochondria damage [31].
In our previous data, an increase level of nitrite, a stable product
of NO, was detected in the culture medium of esophageal carcinoma
cells in arsenite-treated apoptosis[32]. Calcium ions (Ca2+)
act as a universal second messenger in a variety of cells. Numerous
functions of all types of cells are regulated by Ca2+ to
a greater or lesser degree. Because of the importance of Ca2+
in biology, numerous methods of analyzing cellular Ca2+
activity have been established. Confocal laser scanning microscopy (CLSM)
allows the precise spatial and temporal analysis of intracellular Ca2+
activity at the subcellular level. This optical technique has
enabled scientists to document the dynamic changes of intracellular
Ca2+ in vitro [33].
Arsenic
may generate reactive oxygen species to exert its toxicity, which is
implicated in DNA damage, signal transduction and apoptosis. What we
are interested in is to see if NO and Ca2+ are involved
in arsenic-induced apoptosis and to observe the changes of its
target organelle—mitochondria. This study is to investigate which
are the original messengers that initiate apoptosis and to detect
quantitatively Ca2+ and NO in the apoptotic process of
esophageal carcinoma cell line induced by As2O3.
MATERIALS
AND METHODS
Cell
line generation and cell culture
The esophageal carcinoma cell line (SHEEC1) was a malignantly
transformed cell line of human embryonic esophageal epithelium
induced by HPV18 E6E7 in synergy with TPA
(12-O-tetradecanoyl-phorbol-13-acetate)[34]. Cells were
cultured in 50ml flasks and 24-well plate (Corning) with serum-free
medium. The culture medium contained of the basal medium (MCDB151)
with trace elements (M-6645 Sigma) and added transferrin,
hydrocorticosone, epidermal growth factor (EGF), insulin (Sigma
Chemical Co.) and extracts of bovine hypophysis (Gibco, BRL), but
without calf serum, nitrite and nitrate, while containing
streptomycin and penicilline (50mg·L-1 for each).
The
administration of arsenic
Arsenic trioxide (As2O3) obtained from Sigma
Chemical Co. (St. Louis MO, Lot A 1010)at concertrations of 0, 1, 3
and 5μmol·L-1 was added into the culture flasks and 24-well
plates, respectively, for 0, 2, 4, 8, 12 and 24 h. The experiment
was repeated once.
Transmission
electron-microscopy (EM) examination
At the endpoints of As2O3 (24h), cells of each
group were digested with 0.25% trypsin, centrifuged, fixed with 2.5%
glutaraldehyde, and routinely prepared for electron microscopic
examination. The samples were observed under transmission electron
microscope
(Hitachi 300).
Cell
cycle and apoptotic rate analyzed by flow cytometry (FCM)
Cells
of repeated experiment were harvested to measure the ratio of
apoptotic cells to survived cells. The cells were washed twice with
PBS, dispersed, and filtered through 360 mesh nylon net to make a
single cell suspension. It was fixed with 700mL·L-1
precooled alcohol in ice. Before analysis cells were suspended in
PBS and stained with propidium iodide. 1×109 cells·L-1
were detected by flow cytometry (FACsort, B-D Co,USA). The DNA
histogram was drawn according to the fluorescent intensity value of
104 cells.
Procedure
of NO detection[35]
The nitrite/nitrate colorimetric method, using the kit purchased
from Boehringer Mannheim Co, was used to detect NO in culture
medium. The culture medium of 0.2 mL from flask was regularly
deactivated at 80℃
for 5 min and deproteinated by centrifugation in 12000 r·min-1
for 30min, and the supernatant was determined. The procedure for NO
determination was as follows: sample solution of 100μL, 50μL
of nicotinamide adenine dinucleotide phosphate (NADPH) and 4μL
of the enzyme nitrate reductase (NR) were placed in a 3mL test tube,
mixed, incubated for 30 min at room temperature, and added 50μL
color reagent I & II, respectively, mixed,and allowed to stand
in the dark at room temperature for 10 to 15min. The NO content of
the samples and the blank was estimated with Shimadzu UV/120
spectrophotometry byλ450nm and was calculated by calibration
curve of standard addition method. The standards were prepared from
known amounts of stock NO3 and NO2 and run in
parallel with test samples in each assay.
Determination of intracellular calcium level using CLSM[33,
36]
The cells were cultured on the coverslips within the glass bottom of
a small cultured dish (No. 0, uncoated, and irradiated. MatTek Co.,
USA). At the exponential growth period, the cells were stained with
10μmol·L-1 fluo-3/AM (Molecular Probe) for 30min at
37℃,
and washed with 135 mol·L-1 NaCl, 10 mol·L-1
HEPES, 0.4 mol·L-1 MgCl2, 1mol·L-1
CaCl2, 1g·L-1 D-glucose, 1g·L-1
bovine serum albumin, pH 7.3 at least 3 times. Then the cells were
placed in the culture medium 199 to maintain them in living state.
Before and after administration of As2O3 the
fluorescence intensity was determined by CLSM in dynamic changes for
up to 900 s. Using scan-time series menu, time series was used to
scan some definite cells repeatedly to monitor the dynamic changes
in fluorescent intensity of intracellular Ca2+ content
over time. The parameters of the CLSM (Ultima 312, Meridian
Instruments Inc., USA) were as follows: the excited light 488nm, the
emission light 530 nm and pinhole 10-40nm. The fluorescent intensity
of pixel was collected and managed with the software of the
instrument.
RESULTS
Cell
apoptosis
Ultrastructural morphological changes of mitochondria in As2O3
treated cells were described in the previous reports[25,26].
Cells treated with As2O3 at different
concentrations for 24 h displayed an apoptotic appearance. Under
electron microscope, condensed and marginated chromatins in most of
the nuclei appeared accompanying swelling mitochondria (Figure 1).
By flow cytometry, time course study on As2O3
induced apoptosis revealed that apoptotic peak can be observed as
early as 12 h after the incubation of arsenic trioxide in 3μmol·L-1.
The apoptotic cells accounted for 5.0% of total cell population at
12 h and 28.3% at 24 h (Figure 2).
Figure
1 Ultrastructure
of SHEEC1 cell treated with 3μmol·L-1 As2O3.
Apoptotic appearance displayed with swelling of mitochondria and
nuclear chromatin coagulating and merginating. m, mitochondria. n,
nucleus. EM, ×7000
Figure
2(PDF) DNA
histogram of SHEEC1 cell in 24 h after 3μmol·L-1 of
As2O3 adding. ap, apoptotic peak.
NO
determination
When As2O3 acted on the SHEEC1 for 2-24h, in
0, 1, 3 and 5μmol·L-1 As2O3,
NO in cultured medium was increased at the time points. The amount
of NO released from SHEEC1 was increased from the basal condition
(0.98-1.00 ×10-2μmol·L-1) up to the
high level (1.48-1.52 ×10-2 μmol·L-1)
(8h) and maintained for 24h (Figure 3). The concentration of NO in
different groupsvaried, high concentration of NO in 5μmol·L-1
of As2O3 and low concentration of NO in 1μmol·L-1
of As2O3.
Dynamic
change calcium of intracellular calcium
To show the time course of changes in Ca2+ in individual
cells, the changes in fluorescence intensity (arbitrary unit, au) at
different representative cells were measured. Upon the initiation of
stimulation by As2O3, all the cells responded
with a rapid rise in [Ca2+] from 1.00 au. to 1.09-1.38 au
of fluorescent intensity. The peak levels of Ca2+ in all
cells were consistently reached at about 900s after stimulation
(Figure 4A). In the control group, without being treated with As2O3,
the fluorescent intensity of cell, were remained on the basal line
(Figure 4B).
Figure
3(PDF)
NO determination of SHEEC1 treated with different concentration of
As2O3. NO increased markedly in 5μmol·L-1
of As2O3 group (A), intermediately in 3μmol·L-1
of As2O3 group (B) and lowly in 1μmol·L-1
of As2O3 group (C). The control group, 0μmol·L-1
of As2O3, were remained on the basal lines
(D).
Figure
4(PDF)
Dynamic changes of intracellular calcium in 7 cells of SHEEC1
treated with As2O3. A, SHEEC1 cells treated
with As2O3 in 3μmol·L-1. B,
Control group without adding As2O3.
DISCUSSION
In
general, the process of cell apoptosis involved three phases: the
initiation phase, the effector phase and the degradation phase [37].
The initiation (or signal transduction) phase is the stage in which
specific or non-specificpro-apoptotic signal transduction pathways
are activated. The effecter (or central control) phase mainly occurs
in the mitochondria[38] where mitochondria membranes are
unstable as a result of the action of the permeability alternation.
Some genes such as p53 and bcl-2, activate in this phase[39-41].
The degradation (or morphological and biochemical changes) phase
manifest the postmitochondrial features of apoptosis, in which
soluble intermembrane proteins released from mitochondria played an
active role in the activation of proteolytic destruction. In our
previous reports, we investigated the early changes of the apoptotic
cells induced by As2O3 and defined the phase
in which As2O3 was involved[26, 27].
Our
results demonstrated that As2O3 acted directly
on mitochondria for the early stage of apoptosis. The alteration of
mitochondria in arsenic trioxide treated tumor cells could be
observed as early as 2 h after the treatment[27, 30] . In
this study we investigated signal messengers of apoptosis, by first
selecting both messengers of NO and Ca2+ in the apoptotic
pathway.
Experiments
on the effects of various modulators (dose and time lag) of arsenic
in the level of Ca2+ and NO were carried out. Nitric
oxide (NO) is a free radical generated in cells by nitric oxide
synthases (NOS)[42]. It is a gaseous inter- and
intra-cellular messenger that plays as a signaling molecular in many
physiological and pathological processes and it is also a cytotoxic
agent involved in many diseases, whichhas been elaborated
extensively during the last decade. Various intra- or extra-cellular
factors act on mitochondria to produce NO. NO binds to cytochrome
oxidase[43], blocks respiratory chain and induces
apoptosis[44, 45].
Cells
themselves control intracellular Ca2+ concentration ([Ca2+])
strictly with several Ca2+ regulatory mechanisms, such as
Ca2+ channels, Ca2+ pumps, and Ca2+
exchangers. The role of calcium is as the important intracellular
signal element in regulating cell death[46]. As revealed
by previous reports, it seems that calcium changes in apoptosis vary
with stimuli and cell lines[47]. This data suggested that
an early, gradual and sustained increase in intracellular Ca2+
is necessary for the appearance of apoptotic characteristics. In the
examination of CLSM with Fuo-3 AM as a calcium indicator, we found
that a rise in intracellular calcium was elicited at once after
application of As2O3. The mechanism of how
arsenic increases intracellular calcium levels was not clear at this
moment. Arsenic has been shown to disrupt mitochondria and may
elevate intracellular calcium via a signal transduction pathway.
Arsenite has also been reported to activate protein kinase C and
mitogen-activated protein kinase[48]. These kinases are
known to be involved in the calcium signal transduction pathway.
According
to the previous reports, the relationship between NO, Ca2+
and mitochondria in apoptosis is as follows:various extracellular
factors can induce the increase of intracellular Ca2+
levels ([Ca2+]i), modulating cellular
signaling and gene expression, and the increased ([Ca2+]i)
effect on NO production through the iNOS pathway[49, 50];
mitochondria are a source of NO[51], the production of
which may affect energy metabolism, O2 consumption and O2
free radical formation[52]; mitochondrial Ca2+
uptake in combination with NO production triggers the collapse of
mitochondrial membrane potential, affecting mitochondrial
respiration and culminating in delayed cell death[53].
In
conclusion, our data proved that increased calcium ions and nitric
oxide triggered by As2O3 may play an important
role in arsenite-induced apoptosis in esophageal carcinoma cells.
The demonstration of the involvement of Ca2+ and NO in
arsenite-induced apoptosis suggests a new direction for studying the
apoptotic pathway.
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