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Introduction
Spider venoms are biochemically and biologically well studied world-wide
and we know as much about spider venoms as we know about other animal
venoms, such as snake venoms, bee venoms, and scorpion venoms. In
general, the main toxin components of most spider venoms have been
well identified as a complex mixture of proteins, polypep-tides,
polyamine neurotoxins, nucleic acids, free amino acids, monoamines,
and inorganic salts that cause a wide range of effects in both vertebrates
and invertebrates[1-3].
As far as the pharmacology and biochemistry of spider venom is
concerned, spider venom represents a wide variety of ion channel
toxins, novel non-neurotoxins, enzymes and low molecular weight
compounds[4].
A number of studies have examined the anti-tumor substances found
in snake venom[5-9], bee venom[10-12], and
scorpion venom[13]. Many toxins isolated from spider
venom have been invaluable in helping to determine the role and
diversity of neuronal ion channels and the process of exocytosis[14-16].
Six peptide toxins (Magi 1-6) were isolated from the Hexathelidae
spider, Macrothele gigas, dealing with sodium channel (Magi
1-5) and high toxicity in lepidoptera larvae of 3.1 nmol/g (Magi
6)[17]. Toxicological assays showed diverse lethal or
paralytic activities of Magi 7-16 in mice and/or insects[18].
The spider Macrothele raveni was indentified as a new species
in the genus Macrothele[19]. There have been two
papers published about raven toxin tests in mice in which the toxins
acted as a neurotoxic peptide and blocked neuromuscular transmission[20,21].
To date there has been no study examining the venom of the spider
Macrothele raveni. In the present study, the effects of M raveni
spider venom on HeLa cells and possible mechanisms of action were
evaluated.
Materials and methods
Reagents RPMI-1640 (Gibco Laboratories, Grand Island, NY,
USA) dissolved in double distilled water with the pH value adjusted
to 7.0 using NaHCO3, was disinfected and stored at -
20 ºC. Fetal calf serum was purchased from Sijiqing Biological
Engineering Company (Hangzhou, China), sterilized, and stored at
-20 ºC. [3H]TdR was purchased from the
Atomic Energy Institute of China (Beijing, China). Verapamil, streptomycin,
penicillin and 0.5% hydrocortisone were purchased from North China
Pharmaceutical Company (Shijiazhuang, China). Propidium iodide (PI),
bovine serum albumin (BSA), and Triton X-100 were purchased from
Sigma Company (St Louis, MO, USA). The rabbit polyclonal antibody
against caspase-3 and rodamine-labeled secondary antibody were purchased
from Santa Cruz Biotech (Santa Cruz, CA, USA).
Spider venom Pure spider Macrothele raveni venom
was collected by electrical stimulation of 15 spiders M raveni
(the weight of each spider was approximately 60 g)[22].
Spider venom was dissolved in phosphate buffered saline (PBS) and
centrifuged at 8000×g for 10 min to remove insoluble
materials. The concentration of spider venom was adjusted to 0 (control
group), 10, 20, 40 mg/L. The spider venom was freeze-dried and stored
at -80 ºC until required.
Cell culture The human cervical carcinoma cell line HeLa
was obtained from the Cellular Biology Institute of the Chinese
Academy of Sciences (Shanghai, China). The frozen cells were defrosted,
transferred into the culture medium RPMI-1640 supplemented with
10% fetal calf serum, 100 kU/L streptomycin, and 100 kU/L
benzyl penicillin. The cells were grown at 37 °C under a humidified
atmosphere of 5% CO2 .
Determination of apoptosis Morphological evidence of apoptosis
was obtained using acridine orange-ethidium bromide (AO/EB) staining.
Monolayer cell cultures in 96-well plates were used for
these studies. After removal of the incubation medium, the cells
were rinsed and treated with a solution composed of AO/EB
(100 mg/L PBS of each dye). Cells were examined using
fluorescence microscopy and photographed (Olympus, Tokyo,
Japan). Viable cells were colored green with intact nuclei. Non-viable
cells had bright orange chromatin. Apoptosis was demonstrated
by the appearance of cell shrinkage with condensation and
fragmentation of nuclei. Apoptotic cells were easily
distinguished from necrotic cells because the latter
appeared orange with a normal nuclear structure. This
procedure was used to quantify the number of apoptotic
cells after treatment with spider venom. Apoptosis was
also quantified using an enzyme-linked immunosorbent assay (ELISA)
kit (Bindazyme; the Binding Site, Birmingham, UK) that measured
the amounts of mono- and oligo-nucleosomes produced in
the cytoplasmic fraction of lysed cells as a consequence of
DNA fragmentation[23]. After treatment with spider venom,
cells were collected and lysed according to the manufacturer's
instructions. After centrifugation at 11 000×g
for 15 min, equal amounts (0.3 µg of protein) of
the cytosolic fractions were used for the assays.
Specimens for electron microscopy HeLa cells in the exponential
phase were used and cultivated with various concentrations of spider
venom for 24 h. The cells were harvested and fixed with 25 mL/L
glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 2 h at
4 °C. For scanning electron microscopy (SEM) examination, the
specimens were postfixed for 1 h in 2% OsO4, dehydrated
by adding a series of graded ethanol solutions to the
filtration system and then slowly dried over the course of
24 h by evaporation. The filter was removed from
the filtration apparatus and mounted on an aluminum stub, after
which the cells were gold sputter coated. Specimens were examined
with a STEREOSCAN 260 SEM at 25 kV. For transmission electron microscopy
(TEM) examination, cells corresponding to each population were
collected in Haemoline (BioChem Pharma, Allentown, PA,
USA), transferred to microcentrifuge tubes, pelleted,
and fixed in 1% OsO4 (in distilled H2O).
A total of 4×107 cells were sorted to collect 2×106
cells representative of each of the individual populations. After
dehydration through a series of graded alcohol and propylene
oxide solutions, the cells were infiltrated with Epon
(epoxy resin) and polymerized. Ultra-thin sections were
cut, recovered on Formvar-coated copper grids, stained
with uranyl acetate and lead citrate, and then examined
with a 100 CXII transmission electron microscope (Jeol,
Tokyo, Japan) operated at 80 kV.
Cell proliferation assay The inhibition of [3H]TdR
incorporation into DNA was examined using the pulse-labeling method.
Proliferating HeLa cells were seeded onto 96-well plates and incubated
for 16 h. HeLa cells were treated with different concentrations
of spider venom (0, 10, 20,and 40 mg/L) for the next 24, 48,
or 72 h. [3H]TdR (37 MBq/L) was added and HeLa cells
were exposed to [3H]TdR for 16 h. The cells were then
washed three times with PBS, lysed with 1 mol NaOH. After that the
cells were harvested onto glass fiber filters with an automatic
harvester. Filters were dried and radioactivity was quantified using
liquid scintillation counter (Beckman LS6500, Fullerton, CA, USA).
Results were expressed as the percent of specific lysis. Percent
of specific lysis=100×[percent released from the target in
the presence of effectors (experimental release)-percent released
from the target in the presence of medium only (spontaneous release)]/(maximal
release-spontaneous release).
Cell cytotoxicity assay Lactate dehydrogenase (LDH) release
from cells was used as an index of cytotoxicity or necrosis. The
quantity of LDH released by the cells into the medium was measured
by the decrease in the absorbance at 340 nm for NADH disappearance
within 5 min[13]. After incubation with various concentrations
of spider venom (0, 10, 20, and 40 mg/L) for 24 h, the cell
culture supernatant and medium (100 mL) were mixed with 900 mL PBS
and 20 mg/L BSA. The percentage of LDH release (n=6) was
equal to LDH activity in the medium divided by activity in both
the cell culture supernatant and the medium×100%.
Cell cycle distribution using flow cytometry Bivariant flow
cytometry was carried out on cells grown in the presence (control
group) or absence of spider venom (10, 20, and 40 mg/L) after 24
h. Cells were collected and washed in cold PBS twice and resuspended
in 100 mL of binding buffer (HEPES containing 2.5 mmol/L CaCl2).
Fluorescein-labeled annexin V and PI were added to the cell suspension.
Cells were then analyzed using flow cytometry (Becton Dickinson,
Mountain View, CA, USA). The DNA content of the cells was determined
by staining with PI. The cells were incubated in 100 mL of fixing
solution (PBS containing 4% formaldehyde) for 15 min at 4 ºC,
washed in PBS, resuspended in permeabilizing solution (PBS containing
0.1% saponin and 0.1% sodium azide) in the presence of 10 mL of
PI, and incubated at 4 ºC for 15 min. The cells were then washed
with PBS and immediately analyzed using flow cytometry.
Western blot analysis HeLa cells exposed to different
concentrations of spider venom ( 10, 20, 40 mg/L and control) were
incubated in six-well plates at a density of 2.5×105
cells/well for 48 h, followed by a further cultivation of 48
h with the culture medium replaced with 1 mL of serum-free RPMI-1640.
The serum-free medium was totally collected. Equal amounts of proteins
in each sample were resolved in 10% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and the proteins were transferred
onto nitrocellulose membranes. After being washed in 10 ml/L
fat-free milk, the membranes were incubated with the appropriate
dilution of rabbit polyclonal caspase-3 antibody and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) antibody. The membranes were then incubated
with a horseradish peroxidase-conjugated secondary antibody. Proteins
were detected using an enhanced chemiluminescence (ECL) kit according
to the manufacturer's protocol (Amersham, Amersham, Buckinghamshire,
UK).
In vivo reduction in tumor size in nude mice using spider
venom We induced cervical tumors by injecting HeLa cells (200 µL)
under the skin of nude mice with a concentration of 4×109
cells/L. Three weeks after tumor growth was established, treatment
was initiated. Nude mice with tumors were randomly placed into three
groups (10 mice/group) and treated with spider venom, 1.0, 2.0,
4.0 µg/g body weight of mice three times per week for 3 weeks
by tail vein injections. The control group of 10 mice received distilled
water only as a placebo. The mice were killed and tumor size was
evaluated by caliper measurements, and tumor volume was calculated
as length×width×depth.
Statistical analyses Significant differences between groups
were tested using Mann-Whitney U tests with a significance
level of 95%. Differences between groups were considered statistically
significant at p<0.05. Analyses were carried our using
the Prism software package (GraphPad, San Diego, CA, USA).
Results
Effects of spider venom on viability and apoptosis in HeLa cells
Our study was carried out using HeLa cells grown in serum-free
monolayer cultures. We observed using light microscopy
that treatment with spider venom (10, 20, and 40 mg/L)
initially produced clusters of packed cells (data not shown).
After 24 h of treatment, the bulk of the cells appeared to be seriously
damaged. To investigate the type of cell death induced
by spider venom, the cells were stained with AO/EB, which
allows the identification of viable, apoptotic and necrotic
cells based on color and appearance. Staining with AO/EB
of the samples treated with different concentrations of spider venom
(Figure 1) showed different percentages of orange-stained
cells. Viable cells were green with intact nuclei. Non-viable
cells had bright orange chromatin. Apoptosis was demonstrated
by the appearance of cell shrinkage with condensation and
breaking up of the nuclei. Apoptotic cells were easily
distinguished from necrotic cells because the latter
appeared orange with a normal nuclear structure. This
procedure was used to quantify the number of apoptotic
cells after treatment with spider venom. A number of cells exhibited
a flattened polygonal morpho-logy, whereas other cells
showed morphological features of apoptosis, consisting
of a decrease in cell volume, membrane broken, and breaking
up of the nuclei. Moreover, the majority of these non-viable
cells, together with a number of viable cells, showed broken nuclei
and other signs of apoptosis.
Morphological observations After exposure to spider venom
for 24 h, SEM confirmed that HeLa cells treated with spider venom
had morphological features indicative of apoptosis (Figure 2A,2B).
Compared to cells treated with control supernatants,
approximately 25% of cells exposed to spider venom (20 mg/L)
for 24 h appeared to be markedly reduced in size. These cells were
devoid of the villous projections found on the surfaces
of control cells. In addition, many of the spider-venom-treated
cells exhibited perturbations of their plasma membranes
as evidenced by membrane smoothing. Ultrastructural characterization
of cells representative of the individual populations was achieved
through cell sorting and subsequent evaluation using
TEM. Cells treated with spider venom possessed well-defined
plasma membranes and contained intact organelles with no evidence
of nuclear condensation (Figure 2C). In contrast, the majority
of cells in the venom-treated group exhibited morphological characteristics
typical of cells in the early stages of apoptosis. These characteristics
included a decrease in cell size, intact cell membranes, and flocculation
of the nucleus (Figure 2D). None of these characteristics were present
in untreated cells.
Effect of spider venom on [3H]TdR incorporation
in HeLa cells Inhibition of DNA synthesis was examined in the
presence of spider venom (Figure 3). Proliferating HeLa cells were
exposed to different concentrations of spider venom for 24, 48,
or 72 h. The cells were then treated with [3H]TdR for
12 h. Synthesis of DNA in these cells was inhibited, with IC50
ranging from 20 to 30 mg/L after 24 h, 48 h, 72 h. The degree of
growth inhibition of HeLa cells in the presence of spider venom
was dose-dependent. Spider venom significantly decreased cell proliferation,
but the degree differed with the variant concentrations (P<0.01).
The spider-venom-induced inhibition of cell proliferation was also
time dependent. Spider venom significantly decreased cell proliferation,
and again the decrease differed with time (P< 0.01).
Cell cytotoxicity of spider venom on HeLa cells Cell cytotoxicity
was directly measured by LDH release (Figure 4). After 24 h
incubation, spider venom (0, 10, 20, and 40 mg/L) significantly
increased cell cytotoxicity (P<0.05) compared with the
control group (5.95%). Increased cell cytotoxicity occurred with
increased concentrations of spider venom. The activity is to be
time- and dose-dependent.
Effect of spider venom on apoptosis , necrosis ratio, and cell
cycle Cells were exposed to different concentrations of spider
venom (10, 20, and 40 mg/L). Cells in the early phase of apoptosis
were labeled using single annexin V fluorescence. The ratio of apoptosis
and necrosis in the cells increased with increasing venom concentrations.
The early stages of apoptosis and necrosis started 5 h after treatment
of HeLa cells with 40 mg/L spider venom (Table 1).
Cell cycle distribution, cell proliferation and apoptotic damage
of DNA were determined using a flow cytometer. Results showed the
accumulation of cells in S and G2/M, and a corresponding
reduction in the percentages of cells in G0/G1
(Table 2).
Effects of spider venom on the activitiy of caspase-3 To
elucidate the pathway leading to apoptosis, we examined the activation
of caspase-3, which was reported to initiate apoptosis after various
stimuli. HeLa cells treated with spider venom (10, 20, and 40 mg/L)
for 48 h were analyzed for enzymatic activity using a Western blot
analysis. Caspase-3 activity changed at 48 h in HeLa cells (Figure
5). The activity was enhanced significantly (1.7-, 2.2-, and 4.3-fold,
respectively, versus controls, p<0.01). Western blotting
analysis, revealed that the exposure of HeLa cells to spider venom
enhanced the activity of caspase-3, which became four-fold
higher than that in the control cells (Figure 5).
In vivo reduction in tumor size in nude mice using spider
venom We have investigated the effect of spider venom in nude
mice with tumors. Mice were killed 21 d after treatment and tumor
size was determined. In the groups injected with various concentrations
of spider venom by the tail vein, the size of the tumor inside the
skin was significantly smaller (P<0.01) than in untreated
mice (Table 3).
Discussion
A number of possible biological and biochemical effects of spider
venom were investigated in the present study.
Cells excluding vital dyes were considered to be viable.
However, our current results suggest that many of these cells are
actually undergoing apoptotic cell death despite having
relatively intact cell membranes. Spider venom at doses
of 40, 20, and 10 mg/L significantly decreased cell proliferation
in HeLa cells, in a dose-dependent and time-dependent manner.
Aside from the inhibitory effect of spider venom on cell proliferation,
spider venom at the same doses (10, 20, and 40 mg/L) significantly
increased cytotoxicity determined by LDH release in the cells. The
cytotoxic effect of spider venom reached a plateau at doses over
160 mg/L. The cytotoxic effect of spider venom could be attributed
to necrosis[24]. The total number of cells present will
be directly proportional to the background-subtracted fluorescence
values, which represent LDH activity. It is relatively easy to distinguish
between complete cell lysis occurring at time zero versus 50% cell
lysis at 24 h, 48 h, and 72 h and this result was
observed in the "lysed"data (Figure 4).
Meanwhile, we directly measured apoptosis using flow cytometry.
Apoptosis in HeLa cells was increased in the spider venom-treated
groups. It is possible that apoptosis contributes to the cytotoxic
effect of spider venom. Changes in cells associated with the early
phases of apoptosis include a loss of cell membrane phospholipid
symmetry. The effect of spider venom on HeLa cells was analyzed
using fluorescein-labelled annexin V-stained and PI-stained cells
(Table 1). Thus, our findings revealed that spider venom resulted
in direct necrosis and indirect apoptosis (Table 1) to kill HeLa
cells.
To examine whether growth inhibition of HeLa cells by spider venom
resulted from cell cycle arrest, cell cycles treated with spider
venom were analyzed using flow cytometry. The inhibitory effect
of spider venom on growth of the cells may result from G0/G1
cell cycle arrest.
Caspases are common and critical components of the cell death pathway.
Among these caspases, caspase-3-like proteases mediate the initiation
and/or execution stages of programmed cell death[25,26].
The active enzyme takes part in the execution phase of apoptosis
and it has been demonstrated that a high level of activity of effector
caspase-3 in tumor cells plays a decisive role in their commitment
to apoptosis[27-30]. Caspase-3 is a key executioner of
apoptosis, whose activation is mediated by the initiator caspases,
such as caspase-9[31]. Spider-venom-induced apoptosis
was preceded by the activation of caspase-3, which was
clearly observed after 48 h of treatment by direct estimation
of its activity and by Western blotting analysis, which
showed the conversion of procaspase-3 to the active form
of the enzyme. This result is the same as norcantharidin-induced
HeLa cell apoptosis resulting from an increase in caspase-3 activity[32].
All these events preceded the appearance of the morphological
signs of apoptosis, which were observed in a large percentage
of cells only after 48 h of treatment (Figure 5). The results
from the present study suggested that the cytotoxic effect of spider
venom on the cells was related to the induction of apoptosis.
Importantly, the anti-tumor in vivo action of spider venom
resulted in either complete or significant regression of the human
cervical tumors established in nude mice. Our in vivo results
demonstrate that spider venom confers a systemic effect of tumor
regression in a xenograft in a dose-dependent manner.
In conclusion, we demonstrated that spider venom caused an inhibition
of cell growth in HeLa cells as a result of cycle arrest and apoptosis.
Moreover, a large part of our study essentially focused on the mitochondrial
pathway and we determined that one action of spider venom was caspase-3
dependent. These new findings suggest that spider-venom-induced
effects may have novel applications for the treatment of cancer.
Acknowledgements
We are indebted to Jun-xin LI ( Research Centre, the Fourth Hospital
of Hebei Medical University) for equipment and technical assistance.
We are also grateful to Ji-biao LI for technical assistance with
the SEM; Jian ZHANG for molecular technical assistance and sharing
his expertise (Hebei Normal University, Shijiazhuang, China). I
thank Dr James P KEHRER (University of Texas at Austin,TX, USA)
for helping us improve the manuscript.
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