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Introduction
Tachyplesin, which is a kind of cationic peptide isolated from the hemocytes of horseshoe crabs, shows antibacterial
activities with similar efficiencies for both gram-negative and gram-positive
bacteria[1]. It consists of 17 amino acid residues
and the structure determined by Edman degradation is:
NH2-K-W-C-F-R-V-C-Y-R-G-I-C-Y-R-R-C-R-CONH
2.
Tachyplesin is very stable in medium because it has a unique structure, which forms a rigid, antiparallel beta-sheet
because of two intramolecular S-S
linkages[1]. So it could be purified from horseshoe crabs hemocytes in a rigorous way. The
cationic nature of tachyplesin interacts with anionic phospholipids present in the bacterial membrane and thereby disrupts
membrane function[2_4].
We previously reported that the peptides could inhibit the growth of several tumor
cells[5]. Li et al found tachyplesin
could induce differentiation of human hepatocarcinoma cell line
SMMC-7721[6]. Chen et al had shown that RGD-tachyplesin
could induce apoptosis in both tumor and
endothelial cells. RGD-tachyplesin activated caspase-9, caspase-8, and caspase-3 and increased the expression of the Fas
ligand, Fas-associated death domain, caspase-7, and
caspase-6[7]. Those studies gave a hint that tachyplesin is a potential
anti-tumor peptide.
Tachyplesin can inhibit cell growth and induce cell apoptosis; however, the precise mechanism has not been elucidated.
A major characteristic of apoptosis was shrinkage of
cells[8]. Activation of K+ channels was an essential pathway in
programmed cell death. The cell-volume decrease was coupled to
K+ release from the
cells[9,10]. In this paper, we investigated if
apoptosis of HL-60 cells induced by tachyplesin was associated with the efflux of cell potassium and shrinkage of cell
volume.
Materials and methods
Preparation of tachyplesin The hemolymph was
collected and tachyplesin was prepared as described in a previous
study[1]. Tachyplesin was solubilized in physiological
saline. The concentration of tachyplesin was determined by the Bradford
method[9].
Cell lines HL-60 cells(human promyelocytic leukemia cells), which grew in RPMI -1640 (Gibico BRL, Grand Island, NY,
USA), containing penicillin100 µg/mL and streptomycin 100 µg/mL and supplemented with 10% fetal bovine serum (SIJIQING
Laboratories, Hangzhou, China).
MTT viability assay HL-60 cells
(1.0×105 /mL) were cultured in 96-well plates (100 µL/well) and treated with 20
µg/mL tachyplesin in the presence or absence of 50 µmol/L z-VAD-fmk, DEVD-fmk, or IETD-fmk (Clontech, 1290 Terra Bella
Ave. Mountain View, CA 94043, USA) for 24 h. In addition, HL-60 cells were treated with 20 µg/mL tachyplesin in the
presence or absence of 5 mmol/L Ba2+ for 24 h. The number of viable cells in each well was estimated by adding 10 µL 0.5
mg/mL MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (Sigma,Louis,MO 63178, USA) solution. The cells
were dissolved with 100 µL of solution that contained 20% SDS and 50% dimethy formamide after cells had been incubated
for 4 h at 37 oC. The optical densities were quantified at a test wavelength of 570 nm and a reference wavelength of 630 nm
using a multi-well spectrophotometer (Bio-Rad Model 540, 2000 Alfred Nobel Drive Hercules, CA 94547, USA). Results were
calculated as the absorbance[5].
Subdiploid DNA analyzed by flow cytometry
HL-60 cells (1.0×106 cells) were treated with 20 µg/mL tachyplesin in the
presence or absence of 50 µmol/L z-VAD-fmk, DEVD-fmk, or IETD-fmk for 24 h. In addition, HL-60 cells were treated with
20 µg/mL tachyplesin in the presence or absence of 5
mmol/L Ba2+ for 24 h. Cells were washed twice in 1×phosphate-buffered
saline and incubated in 1×phosphate-buffered saline containing 100 µg/mL PI, 200 µg/mL RNase after cells were fixed with
70% ice-cool ethanol overnight. HL-60 cells were then analyzed at excitation wavelengths of 488 nm by flow cytometer
(EPICS XL, Coulter Corporation, Fullerton, CA 92834-3100, USA). The percentage of degraded DNA was determined by the
number of cells with subdiploid DNA
divided by the total number of cells examined under each experimental
condition[10].
Analysis of K+ in cells by flow cytometry
HL-60 cells were treated with 20 µg/mL tachyplesin for 2, 4, 6, 8, and 10 h. In
addition, HL-60 cells were treated with 20 µg/mL tachyplesin in the presence or absence of 5 mmol/L
Ba2+ for 24 h. Intracellular potassium concentrations were determined as described in a previous study using flow
cytometer[11]. HL-60 cells treated in the presence or absence of tachyplesin alone or and other reagents were loaded with the potassium-sensitive
fluorescent dye potassium-binding benzofuran isophthalate (PBFI-AM, Sigma) to a final dye concentration of 5 µmol/L for 1
h at 37 °C, 5% CO2 atmosphere prior to examination. Cells were analyzed at excitation wavelengths of 350 nm by flow
cytometry.
Measurement of mitochondrial membrane potential and analysis of cell shrinkage by flow cytometry
HL-60 cells were treated with 20 µg/mL tachyplesin for 2, 4, 6, 8, and 10 h. In additions, HL-60 cells were treated with 20 µg/mL tachyplesin in
the presence or absence of 5 mmol/L
Ba2+ for 24 h. HL-60 cells were loaded with rhodamine
123(2-[6-amino-3-imino-3H-xanthen-9-yl]benzoic acid methyl ester) (Sigma) to a final dye concentration of 10 µg/mL at 37 °C or 15 min, 5%
CO2 atmosphere prior to examination. Cells were examined at the designated time. Mitochondrial membrane potential and size of the
cells were determined by flow cytometry. Cells were analyzed by excitation of the cells containing rhodamine 123 at 488 nm.
The change of fluorescent intensity of rhodamine 123 indicated the change of
mitochondrial membrane potential.
Cells were examined by exciting the cells with a 488 nm argon laser and determining their position on a forward-scatter and
side-scatter dot plot. Light scattered in the forward direction
(180o) was proportional to cell size, while light scattered at a
90o angle (side scatter) was proportional to cell
density[11]. Therefore, as a cell shrinks or loses cell volume, a decrease in the
amount of forward-scattered light was observed, along with a change in side-scattered light.
Data analysis Data were expressed as mean±SD. Statistical significance was evaluated using the Student's
t-test. P<0.05 was considered to be statistically significant.
Results
Change of intracellular potassium
HL-60 cells were
examined by flow cytometry using the fluorescent potassium indicator dye. Intracellular
K+ was detected by PBFI (K+)
fluorescence intensity. The loss of intracellular
K+ was
observed in a time-dependent manner (Figure1).
Loss of mitochondrial membrane potential coupled to the shrinkage of cells
The fluorescent intensity of rhodamine 123 was examined to detect the changes of mitochondrial membrane potential. It was shown that the loss of the mitochondrial
membrane potential of tachyplesin-treated HL-60 cells was in a time-dependent manner (Figure 2). Simultaneously, cells that
decreased in cell volume had a reduced ability to scatter light in the forward direction (180°). Furthermore, these cells also
showed an increase in their ability to scatter light at a 90° angle, indicating an increase in cellular density (Figure 3).
Examinations of each cell size of various groups showed that the loss of cell volume accompanied the changes of
mitochondrial membrane potential in cells.
Different caspase inhibitors protected against DNA degradation but did not prevent the loss of HL-60 cell viability
induced by tachyplesin HL-60 cells treated with tachyplesin for 24 h showed all of the classical characteristics of apoptosis,
including DNA degradation, as determined by an increase in the number of cells with a subdiploid peak of DNA by flow
cytometry (Figure 4).
The presence of 50 µmol/L z-VAD completely inhibited DNA degradation of tachyplesin-treated HL-60 cells (Figure 5F).
Similar results were observed when either 50
µmol/L DEVD (a caspase-3 inhibitor) (Figure 5G) or IETD (a caspase-8 inhibitor)
(Figure 5H) were used, indicating that the concentration of caspase inhibitors used in these experiments were effective in
preventing DNA degradation event.
A significant fall of cell viability was detected by MTT method after HL-60 cells had been treated with tachyplesin for 24
h (Figure 6E). z-VAD, DEVD, and IETD were ineffective in preventing the loss of cell viability in cells treated with tachyplesin
(Figure 6F_H). Interestingly, the loss of cell viability occurred (Figure 6F_H) in the absence of DNA degradation (Figure
5F_H). It was suggested that a cell
viability change was independent of DNA degradation and, in a caspase-independent manner, induced by tachyplesin.
Tachyplesin sensitivity to
Ba2+ The percentage of degraded DNA increased and cell viability reduced when HL-60
cells were treated with tachyplesin (Figures 5 and 6). It was completely inhibited by
Ba2+(BaCl2), which is a blocker of
volume-regulatory K+ channels (Figures 7 and 8). The mitochondrial membrane potential was stable in the presence of
Ba2+ when HL-60 cells were treated with tachyplesin (Figure 9).
This indicated that Ba2+ suppressed the function of tachyplesin. In the
meantime, the concentration of K+ was maintained in HL-60 cells in the presence of
Ba2+ (Figure 10) and the effect of tachyplesin on cell size was eliminated by
Ba2+ (Figure 11). It implied the efflux of cellular potassium ion was blocked by
Ba2+. Ba2+ blocked efflux of the intracellular potassium to maintain cell size and mitochondrial membrane potential. These data
indicated that efflux of the potassium was an important factor for HL-60 cell death induced
by tachyplesin.
Discussion
Bortner and John reported that the loss of intracellular
K+ could prompt cell apoptosis. A loss of intracellular
K+ occurred in the shrunken population of apoptotic cells and the loss of mitochondrial membrane potential was also restricted
to the shrunken population of cells. so they suggested that loss of
cell volume, K+ efflux, and loss of the mitochondrial membrane
potential were tightly coupled[11].
Depolarization of the cytoplasmic membrane was associated with tachyplesin-mediated activity. Matsuzaki
et al's detailed analysis found that the affinity of tachyplesin to the phosphatidylglycerol (PG) membranes was so strong that one
tachyplesin molecule could bind to approx 200 lipid molecules and cause membranes to
leak[4]. Other reports showed that tachyplesin could interact with anionic phospholipid membranes and thereby disrupt membrane
function[2,3]. So tachyplesin can change cell membrane permeability to cause cell death.
The treatment of HL-60 cells with tachyplesin resulted in alterations of intracellular
K+ and the fall of mitochondrial membrane potential in a time-dependent manner, which was coupled to the shrinkage of cells. These results indicated
tachyplesin peptide could interact with cell membranes and affect ion channels. It then led to a leakage of cell
membranes[4].
K+ effused out from the cells at last.
Cell volume is directly related to the movement of ions, with homeostasis being achieved by a balance of osmotic pressure
across the plasma membrane. When the concentration of solute
particles on each side of the membrane is equal, a net
movement of water is inhibited, thus maintaining a constant cell size. Most
cells achieve and maintain this osmotic balance
through the continuous activity of the
Na+/K+ ATPase pump, which creates and maintains an intracellular
environment high in potassium and low in sodium. In
contrast, the extracellular environment typically contains low levels
of potassium and high levels of sodium. Despite the
negative transmembrane potential, a net electrochemical gradient is established
that favors the passive movement of
potassium out of the cell[12,13]. Bortner
et al's results showed
that potassium ion efflux prompted the
movement of water molecules and caused cell-volume shrinkage in cell
apoptosis[14]. In accordance with Bortner
et al's report, it was suggested that the intracellular water would be accompanied with efflux of
K+ to lose in cell apoptosis induced by tachyplesin. Efflux of intracellular water caused cell volume to shrink.
A recent study by Arrebola
et al supported our results that the loss of intracellular potassium occurred in cells
that have lost their mitochondrial membrane
potential[15]. It was reported that the initial stages of apoptosis were
characterized by decreases in
K+. The largest decreases were in mitochondrial membrane potential and occurred before the
release of cytochrome c in U937 cells undergoing UV-induced apoptosis. Shimizu
et al reported that mitochondrial membrane potential
changed and cell death were inhibited if voltage-dependent anion channel were
closed[16]. Bortner et al's study indicated
that decrease of mitochondrial membrane potential was accompanied by mitochondrial
depolarization, an event which follows the onset of the mitochondrial
permeability transition. Various proapoptotic factors such as cytochrome c
and apoptosis-inducing factor were released from
mitochondria to cause cell
apoptosis[14]. Apparently, the decrease of mitochondrial
membrane potential was associated with loss of intracellular potassium. According to our experimental data, loss of
mitochondrial membrane potential was coupled with loss of intracellular
K+ in tachyplesin-induced apoptosis. So loss of
intracellular K+ induced cell apoptosis was related to mitochondrial membrane potential decrease in tachyplesin-induced apoptosis.
HL-60 cells treated with tachyplesin showed DNA degradation, which was the classical characteristic of apoptosis.
Bortner et al's study showed that high extracellular potassium inhibited caspase-3 activation and DNA degradation, and
prevented the loss of cell viability and cell
shrinkage[14]. Efflux of K+ caused the concentration of ions to fall and activate
apoptosis-associated enzymes, including caspases. It is well known that activated caspases could lead to the formation of
DNA degradation and caspase inhibitor can block this process. Our results showed DNA degradation of HL-60 cells was
inhibited by the presence of several of caspase inhibitors, including z-VAD, DEVD, and IETD.
However, it was interesting that these caspase inhibitors did not prevent the loss of HL-60 cell viability induced by
tachyplesin. This indicated that cell death was independent of DNA degradation.
Ba2+ not only inhibited DNA degradation
but also prevented cell death induced by tachyplesin. This means that DNA degradation was inhibited when the efflux of
K+ was blocked by Ba2+. Arrebola
et al's and Bortner et al's results showed
that cell shrinkage occurred early in apoptosis and
the changes of the intracellular K+ preceded apoptotic
changes[14,15]. Bortner et al showed that
activation of the procaspase by dATP and cytochrome c was effectively
inhibited by physiological
K+ concentrations, and an apoptotic
cell from a state of high ionic strength to low ionic strength
permitted both the loss in cell volume and the activation of enzymes
that mediate apoptosis[14]. It gave a hint that efflux of
K+ affected viability of tachyplesin-treatment HL-60 cells prior to the process of
caspase activation and efflux of K+ may prompt cell death in a caspase-independent
manner. Our results showed that tachyplesin had similarly cytotoxic mechanism of decreased intracellular potassium with thapsigargin and calcium ionophore
A23187.
Cell size and the mitochondrial membrane potential of
HL-60 cells was maintained stably in the presence of 5
mmol/L Ba2+. Shimizu et al reported that mitochondrial membrane potential changed and cell death were inhibited if voltage-dependent
anion channel were closed[16]. It was suggested that
Ba2+ could inhibit tachyplesin-induced HL-60 cell apoptosis by blocking
the efflux of K+.
HL-60 cells treated by tachyplesin caused the permeability of cell membranes to change and
K+ effused from the cells. The efflux of
K+ causes water loss in the cells. The volume of cells shrank and caspases were activated. Furthermore, the
changes of K+ in the cells affected the
mitochondrial membrane potential of tachyplesin-treatment in HL-60
cells. Tachyplesin could induce cell apoptosis in a caspase-dependent manner. In addition, tachyplesin can prompt cell death in a
caspase-independent manner because caspase inhibitors did not suppress cell death.
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