Journal of Medical Biomedical and Applied Sciences 5(4): 18-31, 2017
ICV 2015: 34.93
ISSN: 2349-0748
© 2017, JMBAS
Suberoylanilide Hydroxamic Acid (SAHA) with Antioxidant
vitamin E: Induces ER Stress Pathway in Human Cervical Cancer
Cell Line
Monal Sharma1#*, Paromita Gupta1, Madhu Chopra1*, Anil Kumar Mishra2, Aruna Chhikara3
1
Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi-110007, India.
2
Institute of Nuclear Medicine and Allied Science, Brig. S.K. Mazumdar Road, Delhi-56.
India.
3
Department of Chemistry, Dyal Singh College, Delhi University, Delhi-110007, India.
# Current affiliation: Norton Thoracic Institute, St. Joseph’s Hospital and Medical Science 124
West Thomas Road # 105, Phoenix, Arizona 85013
@ Current affiliation: Department of Biological Sciences, St. John's University, Jamaica, New
York, United States of America
*Corresponding Author
1) Dr. Monal Sharma Norton Thoracic Institute
St. Joseph’s Hospital and Medical Science 124 West Thomas Road # 105 Phoenix, Arizona
85013
Email: [email protected]
Highlights:
 HDAC inhibitor induced anti proliferative active with vitamin E.
 HDAC inhibitor induced CHOP expression with antioxidant agent.
 HDAC inhibitor with antioxidant agent induced Reactive oxygen species.
 Catalase enzyme activity decreased after treatment with HDAC inhibitor with vitamin E.
Abbreviation: SAHA, Suberoylanilide hydroxamic acid; HDACi, Histone deacetylase inhibitor; VE, vitamin E; MTT, 3-[4,5dimethylthiazol-2-yl]-2,5diphenyl-tertazolium bromide; DMSO, Dimethyl sulfoxide; ER, Endoplasmic Reticulum
Keywords: Suberoylanilide hydroxamic acid (SAHA); Vitamin E; HeLa; Apoptosis; ER stress pathway; Reactive oxygen species
(ROS)
Abstract: Suberoylanilide hydroxamic acid (SAHA) is a targeted inhibitor of histone deacetylase, which has been reported to
inhibit cell growth, induce apoptosis in a variety of tumor cells and shown to be an anti-cancer agent. This study describes the
anti-tumor effect of SAHA alone and in combination with antioxidant vitamin E in human epithelial cervical cancer (HeLa) cells.
Cell cycle analysis of drug HeLa cells showed slight decrease in cell population at the G1 phase, and increase at the G2/M Phase
with concomitant decrease in S phase. We also observed SAHA induce anti-proliferative effect in a dose dependent manner and
caused DNA fragmentation. Expression of CHOP and Bax/Bcl-2 Ratio were found to be augmented after post treatment. Level of
Reactive oxygen species (ROS) was increased and antioxidant enzyme catalase activity was decreased after treatment, suggesting
that SAHA induced apoptosis is an endoplasmic reticulum stress pathway. This study provides a better understanding of
molecular mechanism of anti- tumor effect of SAHA and gives insight of the development of better undersigned combinatorial
chemotherapy strategies.
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INTRODUCTION
Cancer has traditionally been considered as a disease;
causing genetic defects such as gene mutations and
deletions, as well as chromosomal abnormalities that
result in either the loss of function in tumorsuppressor genes and/or gain of function or hyperactivation of oncogenes [1]. However, there is
growing evidence that gene expression governed by
epigenetic changes is also crucial to the onset and
progression of cancer [2, 3].Over time, an
appreciation of the importance and complexity of
epigenetic events, such as DNA methylation, histone
post-translational modifications, and miRNA
regulation, has generated many new areas of research
including histone acetylation. Being an important
regulator of gene transcription, histone acetylation
has been studied intensively [2]. The enzymes that
regulate histone acetylation are often incongruously
expressed in cancer cells, which can lead to the
silencing of tumor suppressor genes or activation of
oncogenes [3]. The regulatory role played by these
enzymes make them popular targets for cancer
therapy. Histone deacetylases (HDACs) are a group
of enzymes that, in conjunction with histone acetyl
transferases (HATs), regulate the acetylation status of
histone tails. Acetylation of lysine residues on
histone tails by HATs leads to neutralization of their
charge and decrease affinity for DNA [4]. This
“loosening” of the histone-DNA interaction is
associated with conformational changes which allow
for transcription factors to bind to the DNA and
impact gene transcription [5]. HDACs, on the other
hand, remove acetyl groups which lead to a more
compact chromatin conformation that is often
associated with gene repression. Importantly, HDACs
usually do not function alone, but are part of
multiprotein complexes that contain DNA binding
proteins, chromatin remodeling proteins and other
histone modifying proteins that participate together to
regulate transcription [6].
HDACs are categorized into four families, class I, II,
III and IV, based on their structure [7]. Class I
HDACs include (HDACs 1, 2, 3, and 8) are
predominantly localized to the nucleus. Class II
consist of HDACs 4, 5, 6, 7, 9 and 10 and are
detected in both the nucleus and cytoplasm. HDAC
11 is the sole class IV member and resides in the
nucleus [7]. These three classes of HDACs are zincdependent enzymes. In contrast, class III is
comprised of the Nicotinamide adenine dinucleotide
(NAD) dependent deacetylases, sirtuins (SIRT 1 – 7),
which are found in the nucleus, cytoplasm and
mitochondria and have been identified to be involved
in metabolism and aging [8]. Since HDACs are
involved in deacetylating a wide variety of substrates
and have been identified to modulate many cellular
processes they may be used by cancer cells for a
survival advantage. Based on this rationale, efforts to
define which HDACs are involved in cancer
development and progression are being undertaken
[9].
Histone deacetylase inhibitors (HDACi) are a novel
class of small molecules that inhibit the activity of
histone deacetylase enzyme, being evaluated as
epigenetic regulators of gene transcription.
Suberoylanilide hydroxamic acid (SAHA) is a
targeted inhibitor of histone deacetylase, which has
been reported to inhibit cell growth and induce
apoptosis in a variety of tumor cells [10]. Here in this
study, we tried to evaluate anti-tumor effect of SAHA
in human cervical cell lines HeLa. We showed that
SAHA can inhibit tumor growth, induced apoptosis
and increase CHOP expression. The effect of SAHA
on CHOP expression indicates that the ER stress
pathway mediate SAHA induced apoptosis. In
addition we also studied the combinatorial effect of
SAHA with an antioxidant vitamin E on cellular
responses of HeLa, to propose a hypothesis that the
effect of SAHA can be improved by pro-oxidant
property of vitamin E. This study provides a better
understanding of molecular mechanism of antitumor effect of SAHA and gives insight of the
development of better undersigned combinatorial
chemotherapy strategies.
MATERIALS AND METHODS
Cell culture:
The cervical cancer cells were obtained from
National Centre for Cell Science (NCCS, Pune) and
were maintained at 37oC under humidified, 5% CO2
in DMEM supplemented with 10% (v/v) heatinactivated FBS and 1% antibiotics. Suberoylanilide
Hydroxamic acid (SAHA) synthesized in laboratory
was dissolved in DMSO. Vitamin E was obtained
from Sigma-Aldrich Co. and dissolved in DMSO and
added to the growth medium.
In vitro Cytoxicity assay:
Cells viability was determined by using MTT assay.
Cells (5 X 103) were seeded in 96 wells plate.
Untreated and cells treated with varying
concentration of SAHA and VE were incubated with
5 mg/ml MTT for 4 hrs at 37oC. The formazan
crystals were solubilized in DMSO by shaking and
absorbance was measured at 540 nm in micro plate
reader (Tecan, Genious Pro.) Cytotoxicity of
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treatment conditions was expressed in percentage as
follows % Cytotoxicity = 1- (Absorbance of
sample/absorbance of control) X 100
Trypan blue Exclusion Assay:
The loss of membrane integrity in dying cells allows
the preferential uptake of trypan blue, Dead cells
retain the dye while the viable cells exclude and
appear bright. SAHA, VE and SAHA+VE treated
cells were harvested using 1X trypsin/EDTA
(Sigma). 10 µl of the suspended cells was mixed with
10 µl of 0.4% trypan blue (w/v) and counted using
hemocytometer counting chamber.
Cell Morphological Analysis:
The effect of SAHA, VE, SAHA+VE on
morphological change of HeLa cells was assessed by
inverted and phase contrast microscope (Nikon,
Japan) at 40 X magnification.
Cell Cycle analysis:
Untreated cells and cells treated with SAHA, VE and
SAHA+VE were taken and percentage of cells in
G1/S and G2/M phase of cell cycle was determined
by flowcytometry using intercalating DNA
fluorochrome, Propidium Iodide (PI), DNA staining
method [11]. The stained DNA content of cells was
analyzed by FACS CaliburTM (Becton Dickinson,
USA). A minimum of 10,000 cells /sample was
evaluated and the percentage of cells in each cell
cycle phase was calculated using CELLQUEST and
Modfit software.
Analysis of DNA Fragmentation:
Analysis of DNA fragmentation, as indicator of
apoptosis in untreated and drug treated cells was
done by gel electrophoresis method as described in
Ref. [12].
Western Blot analysis:
Expression levels of PARP, caspase-3, caspase-9,
Bcl2, Bax and CHOP proteins were determined by
western analysis, according to the method used in
Ref. [12]. Mouse Monoclonal antibodies against
PARP caspase-3 caspase-9 and CHOP were used
with anti-mouse polyclonal antibody, conjugated to
horseradish peroxidase (HRP). Rabbit polyclonal
antibodies against Bcl2 and Bax were used in
combination with anti-rabbit polyclonal antibodies
conjugated to HRP. Goat polyclonal antibody against
actin was used as equal loading control. The signal
was detected with ECL kit (Pierce).
Detection of cellular Reactive oxygen species:
Endogenous ROS generation was measured by
fluorometric assay with 2′,7′-dichlorofluorescin
diacetate (DCFH-DA). Control and Drug (SAHA and
in combination with vitamin E) treated 7 HeLa cells
were treated with 20µM DCFH-DA dye in dark for
20 mins. The fluorescence intensities (FIs) of the
cells were measured with a Spectrafluor instrument
(excitation, 485 nm, emission, 538 nm; 37°C) in a
96-well fluoroplate.
Effect of SAHA on Antioxidant Enzymes activity:
The activities of enzymes involved in ROS
metabolism, SOD and CAT, were determined in cell
lysates. Cells were resusupended in PBS and cell
lysates were prepared by sonication until the solution
was clear. Superoxide dismutase (SOD), glutathione
peroxidase (GPX) and Catalase (CAT) activities were
measured using the method of Paoletti and Mocali,
method of Flohe & Giinzler and method of Cohen
respectively (ref). Protein concentrations were
determined by Bradford’s method which is especially
recommended for determining the protein content in
cell fractions.
RESULTS
SAHA inhibits human cervical cell growth:
The effect of SAHA and Vitamin E on cell
proliferation was compared in HeLa cell line using
MTT and trypan blue dye exclusion assay. Cells were
cultured alone and with different concentration of
SAHA and vitamin E and combination for 24 and 48
h. The treatment inhibited the cell proliferation of
HeLa cells in a dose dependent manner, as shown in
fig 1. SAHA was able to inhibit 50% cell growth at
concentration 10-4M. Further to test our hypothesis
that Vitamin E (VE) enhances the therapeutic
efficacy of SAHA, cells were treated with SAHA (10 4
M) and vitamin E (10-3M) for up to 24 h (fig 2). Our
data demonstrated that VE alone resulted modest
decrease in cell viability (up to 20%) and significant
growth inhibition when treated with SAHA in a time
dependent manner (fig 2).
Effect of SAHA and VE on morphology:
Morphological changes in the cells with and without
treatment with SAHA and VE were studied using
phase contrast microscopy. Cell shrinkage, rounding
detachment and segregation of cellular structure in
cells were observed when exposed to 10-4 M SAHA
and with 10-3M Vitamin E for 24 h. These
morphological changes suggested that SAHA might
induce apoptotic cell death in HeLa cells. No
morphological changes were observed in Vehicle
control and Vitamin E treated HeLa cells (fig 3).
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Combination of SAHA and VE arrest cell cycle and
induced apoptosis:
The effect of SAHA and VE on the cell cycle of
human cervical cancer cells is shown in Fig. 4. Upon
treatment with either SAHA alone or combination
with VE, the cell cycle distribution of the cells was
markedly changed. The amount of cells in G1 phase
as well as S phase decreased, whereas the population
of the Sub G1 phase and G2/M phase increased. The
cells in sub G1 phase were identified as dead cells.
Apoptosis was quantified to be 22 ± 0.2% and 24 ±
0.4% in cells treated with SAHA alone or in
combination with vitamin E, respectively.
Combination of SAHA and VE causes DNA
fragmentation:
To study the apoptotic response, DNA of HeLa cells
treated with 10-4 M SAHA for 24 h was isolated and
then subjected to agarose gel electrophoresis. A
typical ladder fragmentation pattern (fig 5) was
observed in cells incubated with SAHA or with
combination of vitamin E. No DNA internucleosomal fragmentation was observed in cells
treated with 0.2% DMSO (vehicle control) and
control and cells treated with vitamin E.
Effect of SAHA on apoptosis related proteins:
To elucidate the mechanism by which HDAC
inhibitor (SAHA) and HDAC in combination with
antioxidant VE initiate cell death, we studied the
expression of different proteins by Western blot
analysis. PARP is a nuclear protein known as DNA
repair enzyme and its cleavage is hallmark of
apoptosis [13]. We observed PARP cleavage from
119kDa to 89kDa fragment in SAHA treated and also
in combination with VE treated cervical cells (fig 6
A).
Caspase 3 and caspase 9 are downstream factors
responsible for apoptosis, which in turn are activated
predominately by the death receptor pathway
(extrinsic pathway) and mitochondrial pathways
(intrinsic pathway) respectively. Cleaved caspase-3
and caspase-9 were not detected in any of the treated
cervical cells, suggesting that extrinsic and intrinsic
pathway of apoptosis were not triggered by the
treatment.
B-cell lymphoma-2 (Bcl-2)-family proteins play a
crucial role in the regulation of apoptosis. Bax and
Bcl-2 proteins are hallmarks for 53 mediated
apoptosis [14]. Changes in the expression of cellular
anti-apoptotic proteins, Bcl-2 and pro-apoptotic
protein from the Bcl-2 family Bax were also
examined by immunoblotting in order to determine
whether SAHA induced HeLa cell death by altering
ratio of Bax and Bcl-2. As shown in fig 6 B an
increase in Bax protein was observed when HeLa
cells were treated with SAHA and in combination
with vitamin E. However cells treated with only a
single drug exhibited relatively stable level of antiapoptotic protein Bcl-2. Increase in Bax protein
leading to shift in Bax/Bcl2 ratio, suggests that
apoptosis is favored.
Involvement of CHOP mediated Apoptosis:
CCAAT/Enhancer-Binding Protein Homologous
Protein (CHOP), a member of the C/EBP family, is
induced in response to cellular stresses, especially by
ER stress. CHOP is involved in the process of
apoptosis or programmed cell death associated with
ER Stress. Western blot analysis was done to study
expression of CHOP in single agent (SAHA) and
combination 10 (SAHA + VE) treated HeLa cells.
CHOP protein expression was up-regulated in cells
treated with SAHA and in combination with vitamin
E. Combinatorial treatment seemed significantly
more effective than single drug treatment (fig 6 B).
The result was further confirmed by RTPCR, where
mRNA expression was increased in cells treated with
SAHA and in combination with vitamin E (data not
shown), suggesting that ER stress pathway of
apoptosis might be involved in cell death of HeLa
cells after exposure to SAHA alone and in presence
of vitamin E after 24h.
SAHA induces ROS Production in HeLa Cells:
To explore the apoptotic mechanism associated with
CHOP induction, we investigated the endogenous
production of H2O2 in HeLa cells after post
treatment with SAHA or in combination with VE.
Endogenous H2O2 can be monitored with oxidationsensitive fluorescent probe DCFH-DA. DCF
fluorescence intensity in HeLa cells treated with
SAHA and in combination with VE was twice as
high as in the cells treated with vitamin E indicating
ROS generation in this condition.
Effect of SAHA on Antioxidant Enzymes (SOD,
CAT, GPx) Activity:
Important determinants of cellular antioxidant
capacity are the enzymes superoxide dismutase
(SOD), catalase (CAT), and glutathione peroxidase
(GPX), which are responsible for the elimination of
ROS. Because these enzymes act sequentially to
remove ROS, the balance of the activity of these
enzymes may be as critical in the defense against
ROS as the activity of the enzymes alone. To
investigate whether the HDAC inhibitor; SAHA with
or without vitamin E has any effect on the activities
of antioxidant enzymes. We measured the activities
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of SOD, CAT and GPx in HeLa cells treated with
HDAC inhibitor (SAHA) with or without antioxidant
(Vitamin E). We checked the mRNA expression of
these antioxidant enzymes by RT-PCR. Significant
decrease was observed only on mRNA expression of
CAT with no change in SOD and GPX levels (data
not shown). The activities of SOD and GPX did not
differ significantly between control and SAHA
treated cells, however SAHA with or without vitamin
E significantly decreased the activities of CAT in
comparison to vehicle treated control cells. We
observe 30% and 50% decrease in SAHA and SAHA
with vitamin E treated cells respectively as shown in
Figure 8.
DISCUSSION
HDACi(s) is a novel and promising class of
chemotherapeutic agent that can induce apoptosis and
differentiation, inhibit cell cycle progression and
possess anti-angiogenic and immune stimulatory
properties [15, 16]. Despite numerous studies
demonstrating these activities in a range of tumor cell
lines and animal models, little progress has been
made in understanding the molecular mechanism
underlying their action. A number of HDACi(s) are
currently being tested in early-phase clinical trials
against a variety of cancers and promising results are
being described, supporting the development of these
compounds for clinical Use. In our study, we
particularly tried to understand the molecular
mechanisms of HDACi-induced apoptosis using
hydroxamic acid based HDACi, Suberoyanilde
hydroxamic acid (SAHA).
We examined the effect of SAHA alone and SAHA
in combination with an antioxidant vitamin E (VE)
on growth inhibition and death of HeLa cells with
IC50 = 100 µM (Fig. 1 & 2). We found that SAHA
was effective in suppressing the growth of Hela cells
at this concentration and induced the morphological
changes when used alone and/or in combination with
vitamin E (Fig. 3). This anti-proliferative activity of
SAHA on HeLa cells could be due to or, at least in
part, by the inhibition of DNA synthesis, proliferation
and ultimately by induction of apoptosis. This has
been confirmed by DNA fragmentation assay, which
is considered a hallmark for apoptosis.
SAHA alone and SAHA in combination with vitamin
E treatment showed typical internucleosomal DNA
fragmentation or ladder formation in HeLa cells at 24
h (Fig. 5), indicating that indeed SAHA induces
apoptosis.
The analysis of the cellular DNA content of apoptotic
cells from which degraded DNA was extracted
reveals them as cells with fractional DNA content,
represented by sub G1 peak on DNA content
frequency histogram (11). Currently this approach is
most frequently used to identify the apoptotic cells
using flow cytometry. After 24 h of treatment of
HeLa cells with SAHA and in presence of vitamin E
showed cell population with hypo diploid DNA
content which also confirmed that SAHA causes cell
death by inducing apoptosis (Fig. 4). The results
were similar to that shown by Nihal et al, where
vorinostat in combination with polyphenolic
antioxidant epigallocatechin-3-gallate (EGCG) has
anti-melanoma effect in melanoma cells and showed
significant inhibition of cell proliferation with
increased apoptosis [13].
Caspases are key mediators of apoptosis [17]. Among
the ten distinct caspases, caspase-3 has been reported
to be the executioner as the most downstream in the
apoptotic pathway. Caspase 3 is mainly activated by
many death signals and cleaves a wide range of
cellular proteins with important functions [17, 18].
Moreover it is also reported that caspase-3 is
essential for DNA fragmentation and induces some of
the morphological changes associated with apoptosis
[19]. We did not found any significant changes in the
levels of procaspase -9 or procaspase -3 in SAHA
and vitamin E treated HeLa cells. This observation
suggests that the cellular death caused by these
agents in HeLa cells is probably through
mitochondrial death pathway (Fig. 6). Our results,
together with others [19, 20], support the hypothesis
that caspase -3 mediated apoptotic chromatin
condensation and DNA fragmentation is dependent
on cell types and kind of stimulus.
For example, the MCF7 cell line, which lacks
caspase-3 expression, is known to undergo cell death
in response to stimulation of TNF-α, staurosporine,
and other agents [20].
The Bcl-2 family of proteins serve as critical
regulators of pathways involved in apoptosis [21], the
main protagonists are suggested to be anti-apoptotic
Bcl-2 and pro-apoptotic Bax. The oligomerization of
Bax in the mitochondrial membrane has been shown
to induce cytochrome c release and the subsequent
steps (including caspase-9 and caspase-3) in the
execution phase of apoptosis [22]. HeLa cells treated
with SAHA exhibited reduced levels of Bcl-2
expression with an increased Bax expression (Fig. 6).
Our finding is in line with previous studies showing
in cutaneous T-cell lymphoma where vorinostat
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treatment did not cause any change in Bcl2
expression but instead up-regulated Bax [23]. These
results suggest that the mitochondrial pathway might
be involved in SAHA induced HeLa cell death.
Increase in Bax may be due to translocation of Bax
from cytocol to mitochondria [24]. Gotoh et al
demonstrated that Bax is also crucial in Endoplasmic
reticulum (ER) stress-induced apoptosis. ER is
emerging as a new focal site for the initiation of
endogenous cell death pathways (24). Evidences are
accumulating that ER stress-induced apoptosis is an
important factor in contributing to a variety of
disease, especially in neuro-degenerative diseases,
diabetes mellitus and cancer [25]. Several pathways
based on ER stress-induced apoptosis have been
reported, in addition to the CCAAT/EnhancerBinding Protein Homologous Protein (CHOP)
pathway [26-28]. CHOP has been shown to act as an
inducer of cell cycle arrest and apoptosis during ER
stress [28-30]. In ER stress induced apoptotic
response, the transcription factor CHOP gets induced
at the transcript level, which sensitizes cells to ER
stress by down regulating Bcl-2 [28-30]. To test
whether, cell death induced by SAHA is dependent
on ER stress we checked the expression of CHOP at
protein level. The expression level of CHOP
increased 4.2 folds in cells treated with SAHA alone
and
6.0 folds in cells treated with SAHA in presence of
vitamin E (Fig 6).Taken together, the results for our
study indicated that treatment with SAHA alone and
in combination with antioxidant vitamin E induces
the expression of CHOP, resulting in increased ratio
of Bax to Bcl-2 in human cervical cancer cells.
Oxidative stress is due to a disturbance in the balance
between the production of reactive oxygen species
(ROS) and the efficiency of the antioxidant defense
[31]. In other words, oxidative stress results if
excessive production of ROS overwhelms the
antioxidant defense system or when there is a
significant decrease or lack of antioxidant defense
[31]. Potential biological targets for free radical
attack include lipids, proteins and nucleic acids [32].
Moreover, severe oxidative stress is not only causes
DNA damage and mutations of tumor suppressor
genes, which are initial events in carcinogenesis [31],
but also plays an important role in the promotion of
multistep carcinogenesis [33]. We examined to
confirm whether the mechanism of apoptosis caused
by SAHA in HeLa cells depend on the production of
ROS. Formation of intracellular ROS was evaluated
based on the intracellular peroxide-dependent
oxidation of DCFHDA to form fluorescent
compound DCF. X-Tocopheryl succinate (TOS), a
vitamin E analog, has been shown to induce the
generation of ROS in caspase independent apoptosis
in human lung cancer A549 and H460 cell lines [34].
We observed a significant intensification of peroxide
ions, as indicated by increase in DCF fluorescence in
SAHA treated cells (Fig 7). The rapid formation of
ROS suggests that it is directly produced by SAHA
and is not a byproduct of cell death. Important
determinants of cellular antioxidant capacity are the
enzymes SOD, CAT, and GPx, which are responsible
for the elimination of ROS. To investigate whether
the HDAC inhibitor; SAHA with or without vitamin
E effects on the activities of antioxidant enzymes, we
measured the activities of SOD, CAT and GPx in
HeLa cells. The activities of SOD and GPx did not
differ significantly but the activity of CAT was
considerably decreased in cells treated with SAHA or
in combination with VE (Fig 8). Similarly,
Kachadourian et al. also showed that 2methoxyestradiol could not inhibit SOD activity, but
it does increase superoxide generation in human
leukemia HL- 60 cells. CAT, in turn, protects the cell
from H2O2 generated by various reactions [36]. A
decrease in the activity of CAT could be due to
increase in the lipid peroxidation product,
malondialdehyde or due to exhaustion of the enzyme
because of increased peroxidation [37-38]. Our
findings are also consistent with the other
observations, wogonin, a flavonoid isolated from
Huang-Qin
(Scutellaria
baicalensis),
which
synergistically sensitizes cancer cells derived from
the cervix, ovary and lung to TNF induced apoptosis,
which was associated with inhibition of catalase
activity and an increase of cellular hydrogen peroxide
[39]. We concluded here that SAHA induced ROS
generation and decrease in the activity of catalase
may lead to increase in hydrogen peroxide radicals in
HeLa cells, which play role in CHOP-induced ER
stress apoptosis pathway.
In conclusion, our study umpires the possibility of
combination of SAHA and antioxidant as a potential
chemotherapeutic agent against human cervical
carcinomas. We proposed mechanism of Apoptosis
induced by SAHA with antioxidant in HeLa Cells
(Fig 9) However; further investigations are required
to give more insight in the mechanisms of apoptosis
after treatment with SAHA or SAHA in combination
with antioxidants.
ACKNOWLEDGEMENT
This work was supported by Indian council of
Medical Research (ICMR) fellowship, Government
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of India. MS is recipient of ICMR fellowship. The
support of Department of Biotechnology (DBT)
funding for execution of this work is highly
acknowledged.
Abbreviation: SAHA, Suberoylanilide hydroxamic
acid; HDACi, Histone deacetylase inhibitor; VE,
vitamin E; MTT, 3-[4,5-dimethylthiazol-2-yl]2,5diphenyl-tertazolium bromide; DMSO, Dimethyl
sulfoxide; ER, Endoplasmic Reticulum
with suberonylanilide hydroxamic acid. J. Biosci.
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LEGENDS
Fig 1: Dose dependent cytotoxicity of SAHA. (A:
HeLa cells were exposed to various concentrations of
SAHA and antioxidant (vitamin E) for 24 h; B: HeLa
cells were exposed to various concentration of SAHA
and constant concentration of antioxidant (vitamin E)
for 24h). The result represents the average of three
independent experiments in triplicate ± S.D. p value
less than # 0.05 was considered significant.
Fig 2: The effect of SAHA on the viability of
cervical cancer cells. Cells were seeded in separate
60 mm dish and treated with and without SAHA and
SAHA + VE. After every 6h, cells were counted by
heamocymeter. The result represents the average of
three independent experiments in triplicate ± S.D. p
value of # 0.05 was considered significant.
Fig 3: Effect of SAHA on Morphology of HeLa
cells. Morphological changes of the cells were
examined under phase contrast microscope at 40X
magnification. (A) Vehicle control (B) SAHA (10 4
M) treated cells (C) Vitamin E (10-3 M) treated and
(D) SAHA +Vitamin E treated.
Fig 4: Cell cycle analysis of HeLa cells after drug
treatment. Cells were collected and processed for
flow cytometric analysis of cell cycle distribution.
DNA content was analyzed using PI. (A: Histogram;
B: cell cycle distribution of HeLa cell line after
treatment with vehicle control and various drug
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combinations). The data is expressed as mean S.E.
from twice independent experiments.
for twice different experiments performed in
triplicate. p value of # 0.05 was considered
significant.
Fig 5: DNA fragmentation assay. DNA was
extracted from drug treated HeLa cells and
electrophoresed on 1.5% agarose gel. (S: SAHA
treated cells, V: Vitamin E treated cells, S+V: SAHA
+Vitamin E treated cells, D: Vehicle control and C:
untreated cells (control).
Fig 8: Effect of SAHA on activities of antioxidant
enzymes in HeLa Cells. The cells were treated
SAHA with or without vitamin E for 24 h. The
activity of CAT, SOD and GPx. S: SAHA treated
cells, V: Vitamin E treated cells, S+V: SAHA +
Vitamin E treated cells, D: Vehicle control and C:
untreated cells (control). Each value represents the
mean ± S. E. for twice different experiments. p value
of # 0.05 was considered significant.
Fig 6: Expression of PARP, Procaspase-9, 3, Bax
Bcl-2 and CHOP proteins. Protein was isolated
after the treatment of SAHA alone and in presence of
vitamin E for 24 h and subjected to Western blot
analysis. (S: SAHA treatment; V: vitamin E
treatment; S+V: SAHA + vitamin E treatment; D:
DMSO treatment; C: control).
Fig 9: Proposed Mechanism of Apoptosis induced
by combination of SAHA and Vitamin E in HeLa
cells. SAHA alone and in presence of vitamin E
causes formation of reactive oxygen species (ROS).
High level of ROS cause damage to proteins which
contribute to ER stress and induses CHOP protein, at
transcriptional level, a known key regulator of
apoptosis through ER Stress pathway. The induction
of CHOP thereafter leads to change in bax/bcl2 ratio
and hence causes cellular damage ultimately leading
to
death.
Fig 7: Formation of reactive oxygen species (ROS)
in HeLa cells. The generation of ROS was evaluated
with DCF fluorescence. ROS generation was
expressed as the intensity of DCF fluorescence
incorporated into cells. S: SAHA treated cells, V:
Vitamin E treated cells, S+V: SAHA + Vitamin E
treated cells, D: Vehicle control and C: untreated
cells (control). Each value represents the mean ± S.E.
FIGURES:
FIG 1:
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Journal of Medical Biomedical and Applied Sciences 5(4): 18-31, 2017
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ISSN: 2349-0748
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FIG 2:
FIG 3:
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Journal of Medical Biomedical and Applied Sciences 5(4): 18-31, 2017
ICV 2015: 34.93
ISSN: 2349-0748
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FIG 4:
FIG 5:
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ISSN: 2349-0748
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(A)
(B)
FIG 6:
FIG 7:
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Journal of Medical Biomedical and Applied Sciences 5(4): 18-31, 2017
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FIG 8:
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Journal of Medical Biomedical and Applied Sciences 5(4): 18-31, 2017
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FIG 9:
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