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Cytotoxic effect and possible
mechanisms of Tetracaine on human corneal epithelial cells in vitro
Xin Pang, Ting-Jun Fan
Key Laboratory
for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean
University of China, Qingdao 266003, Shandong Province,
China
Correspondence to: Ting-Jun
Fan. Key Laboratory for Corneal Tissue Engineering, College of
Marine Life Sciences, Ocean University
of China, Qingdao 266003, Shandong Province, China. tjfan@ouc.edu.cn
Received:
2015-10-02 Accepted:
2015-12-25
AIM: To demonstrate the
cytotoxic effect and possible mechanisms of Tetracaine on human corneal
epithelial (HCEP) cells in vitro.
METHODS: In vitro cultured
HCEP cell were treated with Tetracaine hydrochloride at different doses for
different times, and their morphology, viability, and plasma membrane
permeability were detected by light microscopy, methyl thiazolyl tetrazolium
(MTT) assay, and acridine orange (AO)/ethidium bromide (EB) staining,
respectively. Their cell cycle progression, phosphatidylserine orientation in
plasma membrane, and mitochondrial membrane potential (MTP) were assessed by
flow cytometry. DNA fragmentation, ultrastructure, caspase activation, and the
cytoplasmic apoptosis inducing factor (AIF) and cytochrome c (Cyt. c) along
with the expression of B-cell lymphoma-2 (Bcl-2) family proteins were examined
by gel electrophoresis, transmission electron microscope, enzyme linked
immunosorbent assay (ELISA), and Western blot, respectively.
RESULTS: After exposed to Tetracaine at doses from 10.0 to 0.3125 g/L,
the HCEP cells showed dose- and time-dependent morphological abnormality and
typical cytopathic effect, viability decline, and plasma
membrane permeability elevation. Tetracaine induced phosphatidylserine externalization, DNA fragmentation, G1 phase
arrest, and ultrastructural abnormality and apoptotic body formation.
Furthermore, Tetracaine at a dose of 0.3125 g/L also induced caspase-3, -9 and
-8 activation, MTP disruption, up-regulation of the cytoplasmic amount of Cyt.
c and AIF, the expressions of Bax and Bad, and down-regulation of the
expressions of Bcl-2 and Bcl-xL.
CONCLUSION: Tetracaine above 0.3125 g/L (1/32 of its clinical applied
dosage) has a dose- and time-dependent cytotoxicity to HCEP cells in vitro, with inducing cell apoptosis via a death receptor-mediated
mitochondrion-dependent pathway.
KEYWORDS: Tetracaine; cytotoxicity;
human cornel epithelial cells; apoptosis; mitochondrion
Citation: Pang X, Fan TJ. Cytotoxic effect and possible
mechanisms of Tetracaine on human corneal epithelial cells in vitro. Int J Ophthalmol 2016;9(4):497-504
INTRODUCTION
Human corneal epithelial (HCEP) cells, the uppermost protecting barrier of the
cornea with 6-8 layers of cells, are essential for maintaining corneal
transparency and our vision[1]. Once HCEP cells were damaged by trauma,
infection and drugs, corneal epithelial dysfunction occurred and resulted in
chronic inflammation, cornea ulcerations, edema, turbidity, and even blindness[2-4].
Local anaesthetics, such as
Proparacaine, Lidocaine, Tetracaine, and Bupivacaine, are widely used in ocular
diagnostic and ophthalmic surgery in eye clinic[5-6].
Some topical anaesthetics have been reported to have toxic side effects on
corneal epithelial cells after repeated and prolonged usage[7-9]. Among these, Tetracaine, one of the most frequently used ester-type
anaesthetic agents in ophthalmic surgeries for its fast onset of
action and tissue
penetration, has been
reported to have side effects such as re-epithelialization retardation,
epithelial cell membrane damage and microvillial rarefaction in clinical and
animal studies[8-10]. However, the
detailed cytotoxicity of Tetracaine and its possible cellular and molecular
mechanisms have not yet been clearly clarified
due to the lack of an in vitro model[11].
Recently, the established non-transfected HCEP cell line with normal phenotype[12] and functional potentials in corneal equivalent construction[13-14], makes it possible to study intensively the
cytotoxicity of Tetracaine and its underlying mechanisms in vitro. Here, we aimed to investigate the cytotoxicity of
Tetracaine to HCEP cells and its cellular and molecular cytotoxic mechanisms
using an in vitro model of HCEP cells.
MATERIALS AND METHODS
Materials HCEP cells, from a HCEP cell line established previously in our
laboratory[12], were
maintained and cultured in DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA)
containing 10% (v/v) fetal bovine
serum (FBS; Invitrogen) at 37℃ in 25-cm2 culture flasks
(Nunc, Waltham, MA, USA). Tetracaine
hydrochloride (C15H25ClN2O2; Cas
No.: 136-47-0; Purity>98.0%) was purchased from Tokyo Chemical
Industry (Tokyo, Japan). The 20.0 g/L stock solution of
Tetracaine was prepared with serum-free DMEM medium, and step diluted into
concentrations from 10.0 g/L (clinical applied dosage) to 0.15625 g/L (1/64 of
its clinical applied dosage) dissolved in 10% (v/v) FBS-DMEM/F12 medium before
usage.
Experimental Design HCEP
cells were cultured to logarithmic phase and treated with Tetracaine
at concentrations from 10.0 g/L to
0.15625 g/L. For cytotoxicity evaluation, the cell morphology, viability, and
cell cycle progression was checked by light microscopy, methyl thiazolyl
tetrazolium (MTT) assay, and flow
cytometry (FCM) using propidium iodide (PI) staining, respectively. For
apoptosis verification, the plasma membrane permeability, phosphatidylserine
(PS) orientation, DNA status, and ultrastructure was examined by acridine
orange (AO)/ethidium bromide (EB) double-staining, FCM using Annexin-V/PI
staining, DNA electrophoresis, and transmission electron microscopy,
respectively. For apoptosis signaling pathway postulation, the caspase
activation, mitochondrial transmembrane potential (MTP), and mitochondrial-released
cytoplasmic apoptosis inducing factor (AIF) and cytochrome c (Cyt. c) along
with the expression of B-cell lymphoma-2 (Bcl-2) family proteins was examined by enzyme
linked immunosorbent assay (ELISA), FCM using 5,5’,6,6’-Tetrachloro-1’,1’,3,3’-Tetraethybenzimida
(JC-1) staining, and Western blot,
respectively. In all experiments, HCEP cells cultured in the same medium
without any Tetracaine hydrochloride at the same time point were used as blank controls.
Methods
Light microscopy for growth and morphological observations
HCEP cells were cultured in
a 24-well culture plate (Nunc) in 10% (v/v) FBS-DMEM/F12 medium at 37℃ in a 5% (v/v) CO2 incubator. After the cells grown into
logarithmic phase, the culture medium was replaced respectively with the same
medium containing Tetracaine at concentrations 10.0-0.15625 g/L. The morphology
and growing status of the cells were monitored successively with
a TS100 inverted light microscope (Nikon, Tokyo, Japan).
Methylthiazolyl tetrazolium for cell viability assay MTT assay of HCEP cells exposed to Tetracaine hydrochloride was performed as
described previously[15]. Briefly, HCEP cells in 96-well
plates (Nunc) (1×104 cells per well) were cultured and treated as
described above. At a 2-4h interval, 20 μL of 1.1 mmol/L MTT (Sigma-Aldrich, St. Louis, MO, USA) was added
into the medium and incubated for 4h at 37℃ in the dark. Then 150 μL
of dimethyl sulfoxide (DMSO, Sigma-Aldrich) was added to dissolve the formazan
produced at 37℃ in the dark for
15min, and the 490 nm absorbance was measured with a Multiskan GO microplate
reader (Thermo Scientific, MA, USA).
Double fluorescent staining for plasma membrane
permeability assay AO/EB double-staining of
HCEP cells exposed to
Tetracaine was performed as described previously[15]. In brief, HCEP cells in
24-well culture plate were cultured and treated as described above. Then the
cells were harvested every 1-4h by 2.5 g/L trypsin digestion (1-2min) and
centrifugation (200 g, 10min). After stained with AO/EB solution (100 mg/L AO:
100 mg/L EB=1:1) (Sigma-Aldrich) for 1min, the stained cells
were observed under a Ti-S fluorescent microscope (Nikon, Tokyo, Japan). At least 400 cells were
counted in each group. HCEP cells with red or orange nuclei were designated as
apoptotic cells while those with green nuclei as non-apoptotic cells, and their
apoptotic ratio was calculated according to the formula: “apoptotic rate
(%)=apoptotic cells/(apoptotic cells+non-apoptotic cells) ×100%” with at least
400 cells counted in each group.
Agarose gel electrophoresis
for DNA fragmentation assay DNA agarose gel electrophoresis of
HCEP cells exposed to
Tetracaine was performed following the method reported previously[16]. Briefly, HCEP
cells in 25-cm2 flasks (Nunc) were cultured, treated and
harvested as described above. After washed once with chilled phosphate-buffered
saline (PBS), the genomic DNA was isolated with a Quick Tissue/Culture Cells
Genomic DNA Extraction Kit (Dongsheng Biotech, Beijing, China). The DNA
preparation from each group was electrophoresed on a 1% (w/v) agarose gel (200 mA,
260min), stained with 50 mg/L EB for 10min, and observed with an EC3 Imaging
System (UVP, Upland, CA, USA).
Transmision electron microscopy for ultrastructure characterization HCEP cells in 25-cm2 flasks were cultured, treated with 0.3125 g/L Tetracaine and harvested as described
above. After fixed with 40 g/L
glutaraldehyde and 10 g/L osmium tetroxide successively, the cells were
dehydrated and embedded in epoxy resin. Ultrathin sections were stained with 20
g/L uranyl acetate-lead citrate and observed by an H700 transmission electron
microscope (TEM, Hitachi, Tokyo, Japan).
Flow cytometry analysis The cell cycle progression, PS
orientation and MTP of Tetracaine-treated HCEP cells were
detected and analyzed by FCM as described previously[15]. In brief, HCEP cells in 6-well plates were
cultured, treated with 0.3125 g/L Tetracaine hydrochloride and harvested as
described above. After washed twice with 1 mL PBS, the cells were fixed with
70% (v/v) alcohol overnight at 4℃. Then the cells were stained with PI for cell cycle
assay, stained with annexin V/PI using FITC annexin V Apoptosis Detection Kit I
for PS orientation assay, and stained with 10 μg/mL JC-1 for MTP assay,
respectively. The stained cells were detected by a FACScan FCM (BD Biosciences,
San Jose, CA, USA).
ELISA for caspase activation assay
The activation of
caspase-3, -8, and -9 of Tetracaine-treated HCEP cells was performed as described previously[15].
Briefly, HCEP cells in 25 cm2 flasks were cultured, treated with
0.3125 g/L tetracaine and harvested as described above
every 2h. Whole-cell protein extracts were prepared by lysing 1×106 cells in 500 μL RIPA lysis
buffer (Biotime, Beijing, China) and coated into high-binding
96-well microtitre plates (Nunc), 100 μL per well, at 4℃ overnight. After blocked with 5% (w/v) non-fat milk (BD
Bioscience), the wells were incubated successively with 100 μL
rabbit anti-human caspase-3/8/9 (active form) antibodies (1:500) (Biosynthesis
Biotechnology, Beijing, China) and 100 μL HRP-labelled goat anti-rabbit
secondary antibody (1:3000) (cwBiotech, Nanjing, Jiangsu Province, China) at 37℃ for 2h. A colorimetric reaction was induced by addition of 100 μL
chromogenic substrate (0.1 g/L tetramethylbenzidine, 100 mmol/L acetate buffer,
pH 5.6 and 1 mmol/L urea hydrogen peroxide) for 25min in the dark at room
temperature. Color development was stopped with 50 μL H2SO4 (0.5
mol/L), and the 490 nm absorbance of each well was measured, respectively,
using a Multiskan GO microplate reader (Thermo Scientific).
Western blot analysis The
cytoplasmic amount of AIF and Cyt. c, and expression of Bcl-2 family proteins were
detected by Western blot as described previously[17].
In brief, HCEP cells in 25-cm2
flasks were cultured, treated with 0.3125 g/L Tetracaine
and harvested as described
above every 4h. Whole-cell protein extract was prepared
as described above for expression assay of Bcl-2 family proteins, and
cytoplasmic extract was prepared using mitochondrial and cytoplasmic protein
extraction kit (Sangon biological engineering, Shanghai, China) for
mitochondrion release assay of AIF and Cyt. c. The protein extract from the
same number of cells in each group was electrophoresed by 10% (w/v) SDS-PAGE,
and transferred onto nitrocellulose blotting membranes with a semi-dry blot
system. After blocked with 5% (v/v) nonfat milk, the membranes were incubated
with rabbit anti-human IgG monoclonal antibody to Bad, Bax, Bcl-2, Bcl-xL, AIF,
Cyt. c and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (all in
1:1000 dilution) at 4℃
overnight, and HRP-conjugated goat anti-rabbit IgG monoclonal antibody (1:5000)
for 2h at room temperature, respectively. Finally, the membranes were immersed
in chemiluminescence reagents (Pierce, Rorkford, IL, USA) (0.1 mL/cm2)
and visualized in X-ray films. The optical density of each band was quantified
using ImageJ analysis software (NIH, NY, USA) with β-actin as an internal
control.
Statistical Analysis Each experiment was repeated 3 times
independently. All data were presented as mean±SD and analyzed for statistical
significance by one-way analysis of variance (ANOVA) using SPSS for Windows
version 17.0 (SPSS, Inc., Chicago, IL, USA). Differences to controls were
considered statistically significant when P<0.05.
RESULTS
Morphological Abnormality To evaluate the cytotoxicity of
Tetracaine, the growth and morphology of HCEP cells was first checked by light
microscopy. It was found that HCEP cells treated with 10.0-0.3125g/L Tetracaine
exhibited growth retardation and abnormal morphological changes,
such as cytoplasmic vacuolation, cellular shrinkage, turning round,
detachment from culture matrix, appearance of cytopathic effect (CPE) and
eventually death. Whereas, HCEP cells treated with Tetracaine below
the concentration of 0.3125 g/L showed no obvious morphological changes
compared to controls (Figure 1).
Figure 1
Morphological abnormality of Tetracaine-treated HCEP cells Cultured HCEP cells were treated with
the indicated concentration and exposure time of Tetracaine, and their growth
status and morphology were monitored by light microscopy. One representative
photograph from three independent experiments was shown. c: CPE. Bar: 20 μm.
Viability Decline To
verify the cytotoxicity of Tetracaine, the viability of HCEP cells was then
measured by MTT assay. Results showed that the cell viability of HCEP cells treated
with 10.0-0.15625 g/L Tetracaine decreased significantly (P<0.05 or P<0.01) in a dose- and time-dependent manner, while that of HCEP
cells treated with Tetracaine hydrochloride below the concentration of 0.3125
g/L showed no significant difference to controls (Figure 2A).
Figure 2
Viability decline and cell cycle retardation of Tetracaine-treated HCEP cells A: MTT assay. The cell viability of
Tetracaine-treated HCEP cells in each group was expressed as percentage
(mean±SD) of 490 nm absorbance compared to its corresponding control (n=3). B: FCM with propidium iodide (PI)
staining. G1 phase arrest of HCEP cells exposed to 0.3125 g/L Tetracaine was
shown. The number of HCEP cells in different cell cycle phase in each group was
expressed as percentage (mean±SD) of its total cell number (n=3), respectively. aP<0.05, bP<0.01 versus control.
Cell Cycle Arrest To
postulate the growth retardation mechanisms of Tetracaine, the cell cycle
progression of HCEP cells was assayed by FCM
assay using PI staining. It was found that the number of 0.3125 g/L
Tetracaine-treated HCEP cells in G1
phase increased with time, while that in S and G2/M phase decreased with time,
when compared with that of controls (Figure 2B).
Plasma
Membrane Permeability Elevation To determine the apoptosis
inducing effect of Tetracaine, the plasma membrane permeability of HCEP cells was first detected by AO/EB
double fluorescent staining. Results showed that the plasma membrane
permeability of Tetracaine-treated HCEP
cells elevated with dose and time (P<0.05 or P<0.01), while that of HCEP cells treated with Tetracaine below
the concentration of 0.3125 g/L showed no significant difference to that of
controls. The apoptotic ratio of HCEP cells was shown in Figure 3A.
Figure 3 Plasma membrane permeability
elevation and PS externalization of
Tetracaine-treated HCEP cells A: AO/EB
double fluorescent staining. The apoptotic
ratio was caculated as percentage (mean±SD) of the total number of cells
based on the permeability elevation of plasma membrane of HCEP cells (n=3). B: FCM with annexin V/propidium
iodide (PI) staining. The number of annexin V-positive (PS-externalized) HCEP
cells exposed to 0.3125 g/L Tetracaine in each group was expressed as
percentage (mean±SD) of its total cell number (n=3). aP<0.05,
bP<0.01 versus control.
Phosphatidylserine Externalization To
validate the apoptosis inducing effect of Tetracaine, PS orientation in the plasma membrane of
HCEP cells was then examined by FCM assay using annexin V/PI staining. It was
found that HCEP cells treated with 0.3125 g/L Tetracaine exhibited
significant increase of PS externalization, and in number of annexin V positive
cells (PS externalized cells) increased with time (P<0.01) when compared to that of controls (Figure 3B).
DNA Fragmentation To verify the apoptosis inducing effect of Tetracaine, the genomic DNA of HCEP cells was also
checked by agarose gel electrophoresis. Results revealed that genomic
DNA extracted from HCEP cells treated 2-28h with 10.0-0.15625 g/L Tetracaine
was damaged into a highly fragmented state and typical DNA ladders appeared, while
no DNA fragmentation was found in HCEP cells treated with Tetracaine below the
concentration of 0.3125 g/L even for 28h, which was similar as controls (Figure
4A).
Figure 4 DNA
fragmentation and ultrastructural abnormality of HCEP cells A: 1% agarose gel electrophoresis of
DNA. The dosage and exposure time of Tetracaine are shown
in the top and bottom of each lane, respectively. The dispersed DNA ladders were shown.
Marker, D2000 DNA marker. B: TEM photographs. The dosage and exposure
time of Tetracaine are shown in the top of each photograph. chn: Condensed
chromatin; m: Mitochondrion; mv: Microvillus; N: Nucleus;
v: Vacuoles; apo: Apoptotic body. Bar: 1 μm.
The Ultrastructural Abnormality To confirm the
apoptosis inducing effect of Tetracaine, the ultrastructure of HCEP cells was further examined by TEM.
Results showed that HCEP cells treated with 0.3125 g/L Tetracaine for 4h showed
early apoptotic-like ultrastructural changes, such as structural
disorganization, cytoplasmic vacuolization and mitochondrial swelling. Those
treated for 8h exhibited middle-stage apoptotic-like ultrastructural changes
including advanced mitochondrial swelling, chromatin condensation and
intra-nuclear margination, and a few apoptotic body formation. Those treated
for 12h exhibited late-stage apoptotic-like ultrastructural changes such as cell disintegration and a lot of apoptotic body
formation (Figure 4B).
Caspase Activation To
postulated the triggering pathways of Tetracaine-induced apoptosis, the
activation of caspase-3, -8, and -9 of HCEP cells was determined by ELISA using
antibodies against their active forms. Results indicated that caspase-8 in
0.3125 g/L Tetracaine-treated HCEP cells was activated to a peak value at 6h (P<0.05), caspase-9 in the cells was
activated to its peak value at 8h (P<0.01),
and caspase-3 in the cells was activated continuously during the monitor period
of 12h (P<0.01) (Figure 5A).
Figure 5 Caspase activation and MTP
disruption of 0.3125 g/L
Tetracaine-treated HCEP cells A: ELISA using monoclonal antibodies to
the active forms of caspase-3, -8, and
-9. The activation ratio of caspases in each group was expressed as percentage
(mean±SD) compared to its corresponding control based on 490 nm absorbance (n=3). B: FCM with JC-1 staining. The cell
number of JC-1 positive (MTP disrupted) HCEP cells in each group was expressed
as percentage (mean±SD) of its total cell number (n=3). aP<0.05,
bP<0.01 versus control.
Mitochondrial Membrane Potential Disruption To verify the involvement
of a mitochondrion-dependent pathway in Tetracaine-induced apoptosis, the MTP of HCEP
cells was assayed by FCM using JC-1
staining. Results revealed that the MTP
of HCEP cells treated with 0.3125 g/L Tetracaine was disrupted significantly
with time. The number of monomer JC-1 positive cells
increased from 1.73%±0.53% of
control to 67.63%±3.75% at 4h
and 78.61%±4.24% at
8h, respectively (Figure 5B).
Quantitive Changes of Apoptosis-triggering Proteins To confirm a mitochondrion-dependent pathway is involved in Tetracaine-induced apoptosis, the cytoplasmic amount
of AIF and Cyt. c, and the expression of Bcl-2 family proteins in HCEP
cells were further detected by Western
blot. It was found that the expression level of Bax and Bad was
up-regulated, that of Bcl-2 and Bcl-xL was down-regulated, and the amount of
cytoplasmic AIF and Cyt. c was up-regulated in HCEP cells after exposed to
0.3125 g/L Tetracaine for 4 and 8h, respectively (Figure 6).
Figure 6 Western blots of apoptosis-triggering proteins in 0.3125 g/L Tetracaine-treated HCEP cells A: Western blot
images. The cytoplasmic Cyt. c and AIF, and the expression pattern of Bcl-2
family proteins in HCEP cells were shown. B: Densitometry analysis. The
relative level of protein amount was expressed as percentage (mean±SEM) of
protein band density compared to an internal control of β-actin (n=3). aP<0.05, bP<0.01
versus control.
DISCUSSION
Tetracaine has been reported to have toxicity on rabbit corneal
epithelial cells in vivo[7-8]. However, the cytotoxicity and its underlying mechanisms of tetracaine
are not well understood till now. Here, we investigated the cytotoxicity of
Tetracaine and its apoptosis-inducing mechanisms using an in vitro model of non-transfected HCEP cells for the first time.
To evaluate the cytotoxicity of Tetracaine, the morphology, viability,
and cell cycle progression of HCEP cells were examined by light microscopy, MTT
assay, and FCM
using PI staining, respectively. Our
results showed that Tetracaine at concentrations above 0.3125 g/L (1/32 of its
clinical applied dosage) could induce growth retardation, apoptosis-like
morphological changes with CPE, viability decline, and G1 phase arrest of HCEP
cells in a time- and/or dose-dependent manner. All these indicate that
Tetracaine has a dose- and time-dependent cytotoxicity to HCEP cells in vitro, which has been supported by
previous reports on the cytotoxic effects of Proparacaine, Oxybuprocaine and Lidocaine[15,18-20]. As reported, G1 phase arrested cells will permanently enter a senescent
state that might induce apoptosis
if they could not get through
the G1/S checkpoint[21]. Therefore, the cell cycle arrest, combined with the morphological changes
and viability decrease, imply that tetracaine might have an apoptosis-inducing
effect on HCEP cells in vitro.
As well known, plasma membrane
permeability elevation, PS externalization, DNA fragmentation (also known as
DNA ladder) and apoptotic body formation are all hallmark features of apoptotic
cell death[22-24].
To verify the apoptosis-inducing effect of Tetracaine, the plasma membrane
permeability, PS orientation, DNA integrality, and ultrastructure of the
Tetracaine-treated HCEP cells was then detected by AO/EB staining, FCM using
Annexin-V/PI staining, agarose gel electrophoresis, and TEM, respectively. Our
results displayed that Tetracaine at concentrations above 0.3125 g/L could
induce elevation of the plasma membrane permeability and fragmentation of
genomic DNA of HCEP cells in a dose- and time-dependent manner, Meanwhile,
0.3125 g/L Tetracaine could also induce PS externalization and typical
apoptotic-like ultrastructural changes, such as structural disorganization,
chromatin condensation and apoptotic body formation of HCEP cells. All these
indicate that Tetracaine has an apoptosis-inducing effect on HCEP cells in vitro. The apoptosis-inducing effect
of Tetracaine has also been supported by our previous reports on the
apoptosis-inducing effects of Oxybuprocaine, Lidocaine and Proparacaine[15,18-20].
Generally, apoptosis is triggered two main pathways, a death receptor
mediated extrinsic pathway and an intrinsic mitochondrion-dependent pathway[25], both of which are related
to the activation of different initiator caspases [26]. To
postulate the possible pathways involved in Tetracaine-induced apoptosis, the
activation of caspase-3/-8/-9 of HCEP
cells was characterized by ELISA
using monoclonal antibodies against the active form of caspase-3/-8/-9. We found that Tetracaine could activate
caspase-8, -9 and -3, respectively. As well elucidated, activation of caspase-8
is mediated by death receptor of Fas/CD95 and activation of caspase-9 is
mediated by Cyt. c released from the mitochondrion which is triggered by
disruption of MTP[26]. Our results of the activation of both
caspase-8 and -9 suggest that the Tetracaine-induced apoptosis is most probably
mediated by both a death receptor pathway and a mitochondrion pathway. To
verify the involvement of mitochondrion
in the Tetracaine-induced apoptosis, we then examined the disruption of
MTP by FCM using JC-1 staining,
and found that Tetracaine could induce
MTP disruption of HCEP cells. As known in the mitochondrion-dependent pathway,
the disruption of MTP is a
prerequisite for triggering mitochondrial release of Cyt. c (required for
caspase-9 activation) and AIF (required for caspase-independent initial
chromatin condensation and large-scale DNA fragmentation)[26-27].
Our results indicate that the Tetracaine-induced apoptosis is triggered by a
mitochondrion-dependent pathway. To verify the mitochondrial release of Cyt. C and AIF, we finally examined the
cytoplasmic amount of AIF and Cyt. c along with the expression level of Bcl-2
family proteins by Western blot. We found that Tetracaine could up-regulate the
cytoplasmic amount of AIF and Cyt. c, up-regulate the expression level of Bax
and Bad, and down-regulate the expression level of Bcl-2 and Bcl-xL. As well
demonstrated in Bcl-2 family proteins, anti-apoptotic Bcl-2 and Bcl-xL function
as a gatekeeper of mitochondria to prevent the release of Cyt. c and AIF, while
pro-apoptotic Bax and Bad interact with the mitochondrial permeability
transition pore to induce MTP disruption and release of Cyt. c and AIF from
mitochondria into the cytoplasm[28-29]. Our results of
up-regulation of Cyt. c and AIF,
combined with MTP disruption and caspase-9 activation, indicates that the Tetracaine-induced apoptosis
of HCEP cells is regulated by a mitochondria-dependent pathway. This conclusion
is supported by our previous reports on the apoptosis triggered by local
anaesthetics[15,18-20].
To our
knowledge, this is the first attempt of studying the cytotoxicity of Tetracaine
to HCEP cells and its cytotoxic mechanisms at cellular and molecular levels in vitro. Even these findings are
particularly relevant in deciding the optimal local anaesthetic to be applied
in clinical situations, they do not allow us to predict clinical inferences
directly without further investigations in
vivo.
Tetracaine at concentrations above 0.3125 g/L (1/32 of its clinical
applied dosage) has a dose- and time-dependent cytotoxicity to HCEP cells in
vitro, which is realized by inducing apoptosis in these cells via a death receptor-mediated mitochondria-dependent pathway. Our
findings provide new insights into the non-ignorable cytotoxicity and
apoptosis-inducing effect of Tetracaine on HCEP cells.
ACKNOWLEDGEMENTS
We thank Mr. Ming-Zhuang Zhu, from Key Laboratory of Mariculture, for his
guidance and support during flow cytometry analyses.
Foundation: Supported by National
High Technology Research and Development Program (“863” Program) of China
(No.2006AA02A132).
Conflicts of Interest: Pang
X, None; Fan TJ, None.
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