Key words
trifluoperazine - cytarabine - ifosfamide - neurotoxicity - mitochondria
Introduction
Chemotherapy induced neurotoxicity is the main limitations in cancer patients [1]. Around 30–40% of patients
undergoing chemotherapy experience neurotoxicity, sensory disturbances and symptoms
of pain [2]. The most frequent agents causing
neurotoxicity are chemotherapy agents [2]. A
common mechanism underlying the neurotoxicity is physical damage to the neurons by
chemotherapeutic agent [3]. The physical
damage induced by anticancer drugs leads to mitochondrial damages, oxidative stress,
inflammation, apoptosis, electrophysiological disturbances functional and impairment
in neurons [3]. Chemotherapeutic agents
produce ROS and induce apoptosis in cancer cells [4]. However, ROS produced during cancer therapy may intervene with the
normal tissues and cells and may lead to the various toxic events like
neurotoxicity, nephrotoxicity cardio toxicity and other toxicities. Mammalian nerves
are well-known to be more sensitive to oxidative stress due to their mitochondria
rich axoplasm weak cellular antioxidant defenses and high content of phospholipids
[5]. It has been reported that functional
and structural deteriorations caused by chemotherapeutic agents enhance
mitochondrial free radical production [6].
Oxidative stress caused by mitochondria pathway lead to bioenergetic failure,
depletion of antioxidant defenses, mitochondrial dysfunction, bio molecular damage,
microtubular disruption, neuroinflammation, mitophagy impairment, ion channel
activation, demyelination, and finally neuronal death through apoptosis [7]. Accumulation of dysfunctional mitochondria
due to chemotherapeutic agents increase the vicious cycle of oxidative damage to the
mitochondria and bio molecules that leads a feed-forward mechanism and more
accumulation of ROS and reactive nitrogen species (RNS) in the neurons [8]. In this study, we focused on two main
chemotherapeutic drugs, cytarabine and ifosfamid with neurotoxicity potential.
Ara-C, as first line chemotherapy is used in treatment of hematological cancers
especially in acute myeloid leukemia [9]. A
recent study showed that cytarabine inhibits DNA polymerase γ and leads to
reduction in mitochondrial DNA (mtDNA) content, ROS generation and oxidative damage
in neurons. This study suggests that cytarabine neurotoxicity in neurons originates
in mitochondria and continuous with oxidative stress [10]. Also, IFOS, a structural analog of
cyclophosphamide, is an alkylating chemotherapy agent used for a wide range of solid
and hematologic malignancies [10]. IFOS has
been reported to have adverse neurological effects [11]. Limited works were done to address mechanisms underlying
neurotoxicity of IFOS. It has been suggested that chloroethylamine as an IFOS
metabolite induce the formation of thialysine ketamine which inhibits
electron-binding flavoproteins in the mitochondrial respiratory chain that probably
leads to mitochondrial damages and oxidative stress [12]. Oxidative stress and mitochondrial damages have been reported of
cyclophosphamide as a structural analog IFOS [13].
TFP, a long-established high potency typical antipsychotic drug used in the treatment
of schizophrenia-like and schizophrenia illnesses. Also, TFP as an inhibitor of
calmodulin, can reduce the cellular and mitochondrial Ca2+
overload [14]
[15]. It has been reported that TFP can
interact with the inner membrane of mitochondria, acquired antioxidant activity
toward processes with potential toxicity in cell death, such as ROS formation, lipid
peroxidation of the membrane and MMP collapse and release of cytochrome c [16]. Several studies have also been reported
that TFP, significantly protected mitochondria against the deleterious effects of
Ca2+ and hydrogen peroxide [17]
[18]. The aim of our study was therefore to
explore the effects of Ara-C and IFOS on isolated rat neurons as well as assessing
the protective effects of TFP against Ara-C and IFOS -induced oxidative stress and
mitochondrial damage.
Materials and methods
Animals
Clean grade wistar rats weighting 150−200 g were purchased from
the Pasteur Institute of Iran (Tehran, Iran). This study was approved by the
Research Ethics Committee at the Shahid Beheshti University of Medical Sciences,
and performed strictly in accordance with institutional and international guide
for animal care.
Chemicals
Fetal Bovine Serum (FBS), B-27TM supplement (50X), Neurobasal TM medium, serum
free medium, Dulbecco’s Modified Eagle Medium (DMEM), Trypsin,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
Ethylenediaminetetraacetic Acid (EDTA), β-NGF and L-glutamine
(200 mM) were purchased from GIBICO (Gaithersburg, MD, USA).
Trifluoperazine (TFP), cytarabine (Ara-C) and Ifosfamide (IFOS) was purchased
from Abidi Pharmaceutical Co. (Tehran, Iran). L- carnitine (LC), Sodium
Pyruvate, Glutamine, Hank's Balanced Salt Solution (HBSS), trypan blue,
2′,7′-Dichlorofuorescin Diacetate (DCFH-DA), Acridine Orange
(AO), N-ethylmaleimide (NEM), Rhodamine123, N-(2-hydroxyethyl)
piperazine-N′-(2-ethanesulfonic acid) (HEPES), bovine serum albumin
(BSA), were obtained from the Chemical Co. were purchased from Sigma (Cambridge,
MA, USA).
Isolation of primary rat brain neurons
The rats were sacrificed by cervical dislocation. The brain was removed and put
in HBSS buffer. The blood vessels and pia mater were removed. The brain was
incubated in 0.125% trypsin at 37°C for
10−15 min, rinsed twice with HBSS, and prepared into cell
suspension in DMEM containing 10% FBS. The cell suspension was filtered
through a sterile 70 μm filter, and seeded into DMEM
supplemented with 10% FBS, 1% sodium pyruvate and 1%
glutamine. After 4 h, the medium was replaced with neurobasal medium
containing 2% B27 and 1% glutamine [19].
Cell viability assay
The cell viability was measured by the MTT assay. Cells at 70% confluence
in 6-well plates were incubated for 3 h in normal medium or medium with
different concentration of Ara-C (0–1000) and IFOS (0–1000) or
IC50 3 h Ara-C/IFOS+100 µM TFP. Cells
were collected and stained using trypan blue exclusion dye under optical
microscope at 10x.
Measurement of ROS
The rate of ROS generation was evaluated by using the probe DCFH-DA. In the
presence of ROS, DCFH is oxidized to highly fluorescent dichlorofluorescein
(DCF). Cells at 70% confluence were treated for 1, 2 and 3 h in
normal medium or medium with different concentrations of Ara-C (113, 226 and
452 µM) or IFOS (145, 290 and 580 µM) or IC50
3 h Ara-C/IFOS 100 μM TFP. After the incubation
time, medium was replaced by 10 µM DCFH-DA containing medium,
after 15 min incubation, the fluorescence intensity was measured by
fluorescence spectrophotometer (Shimadzu RF5000U) at the excitation wavelength
of 495 nm and the emission wavelength of 530 nm [20].
Measurement of MMP collapse
The change in the MMP in the isolated neurons were measured by using the cationic
fluorescent dyerhodamine-123. Cells at 70% confluence were treated for
1, 2 and 3 h in normal medium or medium with different concentrations of
Ara-C (113, 226 and 452 µM) or IFOS (145, 290 and
580 µM) or IC50 3 h
Ara-C/IFOS+100 μM TFP. After the incubation
time, medium was replaced by 1 µM rhodamine-123 containing
medium, after 15 min incubation, the medium was removed, and the
fluorescence intensity was measured by fluorescence spectrophotometer (Shimadzu
RF5000U) at the excitation wavelength of 470 nm and the emission
wavelength of 540 nm [21].
Measurement of lysosomal membrane destabilization
The isolated neurons lysosomal membrane integrity was assessed from the
redistribution of the lipophilic dye acridine orange. Cells at 70%
confluence were treated for 1, 2 and 3 h in normal medium or medium with
different concentrations of Ara-C (113, 226 and 452 µM) or IFOS
(145, 290 and 580 µM) or IC50 3 h
Ara-C/IFOS + 100 μM TFP. After the
incubation time, medium was replaced by 5 µM acridine orange
containing medium. After 10 min incubation, the fluorescence intensity
was measured by fluorescence spectrophotometer (Shimadzu RF5000U) at the
excitation wavelength of 470 nm and the emission wavelength of
540 nm [22].
Measurement of lipid peroxidation
Lipid peroxidation was measured by using the thiobarbituric acid assay and
malondialdehyde (MDA) formation. The cells were exposed for 1, 2 and 3 h
with different concentrations of Ara-C (113, 226 and 452 µM) or
IFOS (145, 290 and 580 µM) or IC50 3 h
Ara-C/IFOS+100 μM TFP. After the incubation,
cells were washed with PBS, and then lysed with PBS contain 2% triton.
100 µl of cell lysate was mixed with 200 µl of
thiobarbituric acid (TBA) reagent (containing 3.75% TCA and
0.0925% TBA) and the mixture was incubated at 90°C for
60 min. After cooling, the mixture was centrifuged at 1000×g for
10 min. Calorimetric absorption was measured at 530 nm [23].
Measurement of GSH and GSSG
GSH and GSSG levels in Ara-C/IFOS-treated isolated neurons were measured
by Hissin and Hilf method [24]. After
treatment of isolated neurons with different concentrations of Ara-C (113, 226
and 452 µM) or IFOS (145, 290 and 580 µM) or
IC50 3 h Ara-C/IFOS + 100 μM
TFP, the cells were lysed with 0.5 ml of trichloroacetic acid (TCA)
10% and centrifuged at 11 000×g for 2 min. For
assessment of GSH, supernatant was diluted with phosphate-EDTA buffer and
incubated with 100 µl of the o-phthalaldehyde (OPT) solution for
15 min at room temperature. For determination of GSSG, cells supernatant
was diluted with NaOH 0.1N solution and before incubation with OPT,
200 µl of N-ethylmaleimide (NEM) solution was incubated with
supernatant for 30 min. The fluorescence intensity was measured by
fluorescence spectrophotometer (Shimadzu RF5000U) at the excitation wavelength
of 350 nm and the emission wavelength of 420 nm.
Statistical analysis
All data are presented as the Mean±Standard Deviation (SD) with three
separate experiments. Data were analyzed by GraphPad Prism 5 (GraphPad Software,
La Jolla, CA) using one and two-way analysis of variance followed by post hoc
Tukey and Bonferroni test. P value of less than 0.05 was considered as
statistically significant.
Results
Cell Viability
Cytotoxic effects of Ara-C (0–1000 µM) or IFOS
(0–1000 µM) on isolated neurons showed in the [Fig. 1] a-b. Ara-C and IFOS caused dose
dependent cytotoxicity on the cells and significantly (P<0.05) reduced
cell viability in all used concentrations. The presented data at [Fig. 1] c-d demonstrated that TFP
(100 µM), as a mitoprotective agents prevent of cytotoxicity
induced by Ara-C and IFOS.
Fig. 1 Viability of isolated neurons following treatment with
Ara-C/IFOS for 3 h (a and b) and protective effect of TFP
against Ara-C/IFOS toxicity (c and d). Cell viability determined by
trypan blue assay after incubation of the cells with different
concentration of Ara-C/IFOS. Ara-C and IFOS both decrease neuron
viability in a dose-dependent manner and this decrease is significant at
concentration higher than 100 µM.
(*** p<0.001 vs.
control) (a and b). Protective effect of TFP (100 µM)
are tested against cytotoxicity induced Ara-C/IFOS (c and d). Presented
data showed these agents significantly decreased cell death compared to
treated groups with Ara-C/IFOS alone.
(*** p<0.001 vs.
control, ###p<0.001 vs. treated groups with
Ara-C/IFOS).
ROS production
The effects of Ara-C (113, 226 and 452 µM) or IFOS (145, 290 and
580 µM) on the generation of ROS in isolated neurons are shown
in [Fig. 2] a-b. Ara-C/IFOS has
induced dose and time dependent ROS generation in isolated neurons. When the
isolated neurons were simultaneously treated with Ara-C/IFOS+TFP
(100 µM), the mean fluorescence intensities were significantly
decreased compared to treated groups with Ara-C/IFOS ([Fig. 2] a-b).
Fig. 2 ROS Generation in isolated neurons after incubation with
Ara-C/IFOS for different concentrations and time (1, 2 and 3 h)
intervals and protective effect of TFP against Ara-C/IFOS induced ROS
formation (a-b). Ara-C/IFOS has induced dose and time dependent ROS
production (a-b). TFP, inhibited Ara-C/IFOS -induced generation of ROS
in isolated neurons.
(*** p<0.001 vs. control,
###p<0.001 vs. treated groups with Ara-C/IFOS).
MMP collapse
The effects of Ara-C (113, 226 and 452 µM) or IFOS (145, 290 and
580 µM) on the MMP of isolated rat neurons were presented in the
[Fig. 3] a-b. Ara-C/IFOS
induced statistically (P<0.05) MMP collapse in dose and time dependent
manner. As shown in the [Fig. 3] a-b,
collapse of mitochondrial membrane potential was inhibited after treatment of
neurons with Ara-C/IFOS by TFP (100 µM) at toxic
doses.
Fig. 3 Collapse of mitochondrial membrane potential (MMP) in
isolated neurons following incubation with Ara-C/IFOS for 1, 2 and
3 h and protective effect of TFP against Ara-C/IFOS induced
mitochondrial damages (a-b). Collapse in mitochondrial membrane
potential started 1 h after treatment of isolated neurons with
Ara-C/IFOS at three concentrations. Ara-C/IFOS-induced mitochondrial
membrane potential collapse was time and concentration dependent
(P<0.001) (a). TFP prevented Ara-C/IFOS-induced collapse in
isolated neurons.
(*** p<0.001 vs. control,
###p<0.001 vs. treated groups with Ara-C/IFOS).
Lysosomal membrane destabilization
The effect of Ara-C (113, 226 and 452 µM) or IFOS (145, 290 and
580 µM) showed in [Fig.
4a-b]. Ara-C/IFOS induced statistically lysosomal damages in
dose and time dependent manner. Lysosomal membrane destabilization was inhibited
after treatment of isolated neurons with Ara-C/IFOS by TFP
(100 µM) at toxic doses ([Fig.
4] a-b).
Fig. 4 Lysosomal membrane destabilization in isolated neurons
after incubation with Ara-C/IFOS and protective effect of TFP against
Ara-C/IFOS-induced lysosomal damages (a-b). After 1, 2 and 3 h
treatment, Ara-C/IFOS caused significant (P<0.001) lysosomal
membrane leakage (a). TFP prevented Ara-C/IFOS-induced lysosomal
membrane leakage. (*** p<0.001
vs. control, ###p<0.001 vs. treated groups with Ara-C/IFOS).
Lipid peroxidation
Lipid peroxidation as an indicator of oxidative damage to the lipids was measured
in isolated neurons using MDA assay as a byproduct of lipid peroxidation. We
showed that the amount of MDA as the result of lipid peroxidation significantly
(P<0.05) increased when cells incubated Ara-C (113, 226 and
452 µM) or IFOS (145, 290 and 580 µM) at toxic
dose during 1−3 h ([Fig.
5] a-b). Pretreatment of isolated neurons with by TFP
(100 µM) at toxic doses significantly (P<0.05) decreased
the MDA level after 3 h. incubation time ([Fig. 5] a-b).
Fig. 5 Induction of lipid peroxidation in isolated neurons and
after incubation with Ara-C/IFOS and protective effect of TFP against
Ara-C/IFOS-induced oxidative damages (a-b). Lipid peroxidation
significantly increased when isolated neurons were incubated with
Ara-C/IFOS (a). TFP prevented Ara-C/IFOS-induced lipid peroxidation.
(*** p<0.001 vs. control,
###p<0.001 vs. treated groups with Ara-C/IFOS).
GSH and GSSG content
Isolated neurons were treated with Ara-C (113, 226 and 452 µM) or
IFOS (145, 290 and 580 µM) and 1 h after treatment
decrease in GSH/GSSG ratio were observed. The effects of Ara-C/IFOS on GSH and
GSSG content are shown in [Fig. 6] a-b.
This finding indicates a significant (P<0.001) changes in
GSH/GSSG ratio in concentration-dependent manner. Pretreatment of
isolated neurons with TFP (100 µM) inhibited decrease of
GSH/GSSG ratio. GSH/GSSG ratio significantly (P<0.05)
increased at 1,2 and 3 h following co-treatment with
Ara-C/IFOS+TFP in isolated neurons.
Fig. 6 Effect of Ara-C/IFOS on GSH/GSSG ratio and protective
effect of TFP against Ara-C/IFOS-induced GSH depletion (a-b). As
demonstrated, significant (P<0.001) GSH/GSSG ratio decrease was
found after treatment with Ara-C/IFOS. TFP prevented depletion of GSH
(*** p<0.001 vs. control,
###p<0.001 vs. treated groups with Ara-C/IFOS).
Discussion
The reactive oxygen species are produced in the cell through several pathways. The
generated ROS within cells are rapidly removed by various non-enzymatic and
enzymatic mechanisms [25]. Disruption of the
antioxidant-oxidant equivalence originates oxidative stress and cell damage [25]. The oxidative stress produced by
anticancer drugs cause side effects in the treated patients [26]. When the formation of ROS oversteps repair
capacities, cellular adaptive biological molecules such as proteins, membrane
phospholipids and nucleic acids, become damaged because of oxidative reactions.
Finally, oxidative stress leads to the defeat of normal cellular functions and even
cell death [27]. Methotrexate, Ara-C, and IFOS
are the anticancer drugs that most frequently cause central nervous system (CNS)
toxicity. These agents induce central neurotoxic adverse effects [28]. In the current study, we focused on
cytotoxicity of Ara-C, and IFOS on isolated brain neurons. Our measured toxicity
parameters showed that Ara-C, and IFOS significantly induce ROS formation. Previous
studies showed that Ara-C, and IFOS increase the ROS formation and oxidative stress
in normal and tumor cells [29]
[30]. Also, other examinations showed that
both drugs induce lipid peroxidation and decrease GSH in isolated neurons. Our
results are consistent with previously published data examining Ara-C, and
IFOS-induced oxidative stress in several systems [29]
[30].
Drug-induced mitochondrial toxicity has been investigated well for over 50 years in
academic settings. Drugs may inhibit mitochondrial function in many different ways
[31]. Mitochondrial toxicity has been
identified to cause organ toxicity to the central nervous system, kidney, skeletal
muscle, heart and liver [32]. Drug classes
identified to cause mitochondrial toxicity are anti-diabetic, cholesterol lowering,
anti-depressants, pain medications, certain antibiotics, and anti-cancer drugs [33]. Most of drug-induced mitochondrial
toxicities were not detected in preclinical animal studies. Most of these effects
have been proven through studies in isolated cells and mitochondria [33]. Our results showed that Ara-C, and IFOS
significantly induce mitochondrial membrane potential collapse in isolated rat
neurons, which is consistent with previously published data examining anticancer
drugs-induced mitochondrial toxicity in other tissues.
Lysosomes serve as the cellular recycling center and are filled with numerous
hydrolases that can degrade most cellular macromolecules [34]. Lysosomal membrane permeabilization and
the consequent leakage of the lysosomal content into the cytosol leads to lysosomal
cell death [34]. This form of cell death is
mainly carried out by the lysosomal cathepsin proteases and can have apoptotic,
apoptosis-like or necrotic features depending on the extent of the leakage and the
cellular context [34]. Many lipophilic, weakly
basic drugs accumulate in lysosomes and apply complex, pleiotropic effects on
organelle structure and function [35]. In the
current study we observed lysosomal membrane permeabilization after exposure of
isolated neurons with Ara-C, and IFOS. However, this effect may be due to the
production of reactive oxygen species or mitochondrial damage caused by these drugs
which is known as lysosomal and mitochondrial crosstalk [36].
There are conflicting opinions regarding the administration of antioxidants during
cancer therapy [37]. Antioxidants may reduce
the effectiveness of chemotherapies, which are based on increasing oxidative stress
in tumor cells [37]. For example, Ara-C was
toxic to MLH1 and MLH2 deficient tumor cells, but this cytotoxicity was reduced by
antioxidants [30]. Also, certain researchers
revealed mitochondrial dysfunction and mitotoxicity contribute to amplified
oxidative stress [38]. Therefore,
mitochondrial protective agents may be a good promising strategy form the inhibition
of anticancer drugs toxicity. Mitochondrial permeability transition pore (mPTP)
plays a central role in alterations of mitochondrial structure and function leading
to neuronal injury [39]. Many anticancer drugs
like studied ones in this work, trigger the formation of mPTP, resulting in
increased oxidative stress, impaired mitochondrial respiration function, decreased
mitochondrial membrane potential and release of cytochrome c [40]. Therefore, mitochondrial membrane
permeability inhibitors can block the effects dependent on the opening of the MPT
pore. Inhibition of mPTP has appeared as a promising approach for neuroprotection
and development of well-tolerated mPT inhibitors with favorable blood-brain barrier
penetration is highly warranted [41]. 28
clinically available drugs with a common heterocyclic structure were identified as
mPT inhibitors [41]. In the current study we
tested neuroprotective effect of TFP as a mPT inhibitor [42] against mitochondrial and oxidative stress
induced by Ara-C, and IFOS. Our results showed that TFP as an antipsychotic drug and
inhibitor of mPT reversed all the toxicities induced by Ara-C, and IFOS.
In summary our result confirmed that Ara-C, and IFOS induce cytotoxicity through
mitochondrial dysfunction, oxidative stress and lysosomal damages in CNS neurons.
Also, quite interestingly our data showed that TFP as an antipsychotic drug and
inhibitor of mPT with good penetration through blood-brain barrier can reverse
Ara-C, and IFOS -induced neurotoxicity in isolated neurons.
