Apoptosis inducing properties of 3-biotinylate-6-benzimidazole B-nor-cholesterol analogues

Article abstract of DOI:10.1016/j.steroids.2021.108822

In this work, a series of Biotin-substituted B-nor-cholesteryl benzimidazole compounds were synthesized. The antiproliferative activities of these compounds were evaluated in vitro using a series of human cancer cell lines, including HeLa (cervical cancer), SKOV3 (ovarian cancer), T-47D (thymus gland cancer), MCF-7 (human breast cancer) and HEK293T (normal renal epithelial) cells. These compounds displayed distinct antiproliferative activities against the currently tested cancer cells. The apoptotic properties induced by compound 6d were further investigated. Our results showed that compound 6d could induce the apoptosis of SKOV3 cells, blocking the cell growth in S-phase. Western blotting analyses revealed that compound 6d can induce cell apoptosis via the mitochondria-dependent pathway.

Full text of DOI:10.1016/j.steroids.2021.108822

Steroids 169 (2021) 108822
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Apoptosis inducing properties of 3-biotinylate-6-benzimidazole
B-nor-cholesterol analogues
Zhiling Zhu, Zhiping Liu, Jianguo Cui, Yanmin Huang, Hualong Chen, Yulan Wu,
Xiaotong Huang, Chunfang Gan*
Guangxi Key Laboratory of Natural Polymer Chemistry and Physics, Key Laboratory of Beibu Gulf Environment Change and Resources Utilization, School of Chemistry
and Material, Nanning Normal University, Nanning 530001, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this work, a series of Biotin-substituted B-nor-cholesteryl benzimidazole compounds were synthesized. The
antiproliferative activities of these compounds were evaluated in vitro using a series of human cancer cell lines,
including HeLa (cervical cancer), SKOV3 (ovarian cancer), T-47D (thymus gland cancer), MCF-7 (human breast
cancer) and HEK293T (normal renal epithelial) cells. These compounds displayed distinct antiproliferative ac-
tivities against the currently tested cancer cells. The apoptotic properties induced by compound 6d were further
investigated. Our results showed that compound 6d could induce the apoptosis of SKOV3 cells, blocking the cell
growth in S-phase. Western blotting analyses revealed that compound 6d can induce cell apoptosis via the
mitochondria-dependent pathway.
B-nor-cholesterol
Benzimidazole
Synthesis
Cell-cycle
Apoptosis
1. Introduction
induce HL-60 cell apoptosis at the concentration of 10 μg/mL. Wei and
colleagues have also obtained some B-Nor compounds from Svenzea zeai
that possess good inhibitory activity against Mycobacterium tuberculosis
[10]. In our previous work, we had prepared some different series of B-
Nor steroidal compounds and evaluated their antiproliferative activity
[11–13]. Among these compounds, some B-nor-steroids with cholesteryl
side chain and 6-benzimidazole groups showed significant cytotoxic
activity [14]. These compounds can inhibit cell proliferation by inter-
fering with SKOV3 cell membrane structure and cell metabolic function
[15]. In order to identify the biomolecule that bind to B-Nor choles-
terols, a biotinylated derivative of B-nor-cholesteryl benzimidazole with
considerable bioactivity should be designed and further synthesized.
Using the B-nor benzimidazole as a lead compound, introduced a biotin
group into the 3-position of cholesteryl, leading to the generation of four
Despite the various preventive strategies, early diagnosis and drug
development efforts, cancer is still considered as the second leading
cause of death worldwide [1]. Cholesterol and its derivatives display a
variety of biological functions. As one of the predominant components in
the plasma membrane, cholesterol is found to participate in modulating
the multidrug efflux pumps, contributing to the development of the
multidrug resistance (MDR) phenotype in cancer cells [2]. On the other
hand, cholesterol analogues, such as estramustine sodium phosphate,
exemestane, abiraterone acetate or 2-methoxyl estradiol, have been
developed for treating different cancers. Steroidal compounds are a class
of drug molecules with polycyclic skeleton structure, which play an
extremely important role in maintaining the normal physiological
functions. Modifications on steroidal structure or changes on the steroid
backbone may endow novel biological activities or improve its original
biological function [3–5]. Cholesterol derivatives display anti-cancerous
activities mainly through inducing cancer cell apoptosis and other
cytotoxicity [6–8].
compounds with different benzimidazole rings [16–18].
A non-
biotinylated linker could be incorporated at the suitable position, so
the biological activity of the small molecule could be conserved.
The antiproliferative activity of these compounds in distinct types of
cancer cells was evaluated, and the mechanism of action of the most
potent anti-cancer compound was further evaluated.
B-Nor steroidal compounds can be considered as a class of steroid
derivatives, which are rarely found in the nature. Higuchi and col-
leagues have isolated a B-Nor compound, named Orostanal, from the
Japanese sponge Stelletta hiwasaensis [9]. This compound was able to
* Corresponding author.
E-mail address: ganchunfang2008@126.com (C. Gan).
https://doi.org/10.1016/j.steroids.2021.108822
Received 10 November 2020; Received in revised form 1 March 2021; Accepted 4 March 2021
Available online 12 March 2021
0039-128X/© 2021 Elsevier Inc. All rights reserved.

Z. Zhu et al.
Steroids 169 (2021) 108822
2. Result and discussion
also tested for comparison. The results are summarized in Table 1. It was
observed that, upon linkage of a short chain at 3-hydroxyl, the presence
of an alkyl group on the benzimidazole structure could considerably
affect the biological activity of B-nor-cholesteryl derivatives. Replace-
ment of hydrogen by a methyl group almost abolished this inhibitory
activity. It seems that the presence of an electron-attracting group in the
benzimidazole is more efficient than that with an electron-donating
group. Based on this data, we can find that by the addition of a short
chain in the 3-positon of B-nor-cholesterol benzimidazole derivatives,
the antiproliferative activity of compound 5a, 5c and 5d showed distinct
activity to the test cancer cells, the antiproliferative activity of com-
pound 5b disappeared. In addition, compound 5c (i.e. with electron-
attracting group in benzimidazole structure) showed better biological
activity.
2.1. Chemistry
Generally, biotin can directly connect to the hydroxyl group of ste-
roids by esterification. However, the 3-hydroxy group of cholesterol is
β-configuration, which causes a large steric hindrance due to the 1,4-
interaction between the 3-hydroxy group and the methyl group on the
10-position [17]. At the same time, biotin has a large molecular struc-
ture, so that its carboxyl group is not prone to be directly esterified with
the 3-hydroxyl group of cholesterol. Therefore, a glycol linker on the 3-
position of cholesterol was introduced and then the biotin group was
connected, thereby the purpose of introducing biotin into B-nor-
cholesterol was achieved.
Fig. 1 illustrates the synthesis of biotin-substituted B-nor-cholesteryl
benzimidazole derivatives 6(a–d). In the presence of triethylamine,
compound 1 was treated with methyl sulfonyl chloride to afford com-
pound 2, followed by etherification with glycol to generate compound 3
[19]. Thereafter, compound 3 was ozonized at ꢀ 80℃ and the B-ring of
the cholesterol was broken. Using aluminum oxide as a base, compound
4 was further obtained via aldol condensation. The reaction of com-
pound 4 with different O-phenylenediamine derivatives allowed the
formation of corresponding benzimidazole derivatives 5(a–d). Lastly,
compounds 6(a–d) were yielded by coupling compounds 5(a–d) with D-
Biotin. Their respective structures were confirmed by spectral data.
Reagents and conditions: (a) MsCl/Et3N/DCM, ꢀ 15 ◦C for 5 mins; (b)
Glycol,1,4-dioxane at 90 ◦C for 18 h; (C) O3 at ꢀ 80 ◦C, for 55 mins,
Al2O3, r.t for 12 h;(D) R1, R2-diaminobenzene, EtOH, 65 ◦C for 8 h;(e)
DCC/DMAP/D-Biotin, DCM, 45 ◦C for 12 h.
A series of novel biotinylated compounds derived from B-nor-
Table 1
a
IC50 of compounds against human cancer cell lines. (IC50
,
μ
M).
Compd.
HeLa
SKOV3
T47D
MCF-7
HEK293T
5a
18.19 ±
17.6
16.45 ±
32.8
9.85 ± 8.0
9.21 ±
0.96
23.80 ±
1.38
5b
5c
5d
6a
6b
6c
6d
>100
>100
>100
>100
17.81 ±
1.25
12.42 ±
10.9
13.17 ±
6.5
11.25 ±
6.9
10.39 ±
1.06
28.07 ±
1.42
47.46 ±
11.5
42.43 ±
13.0
50.02 ±
21.3
9.68 ±
0.99
24.22 ±
1.38
24.06 ±
13.0
16.87 ±
3.8
6.88 ± 3.9
19.57 ±
6.0
25.4 ± 34.8
>100
70.69 ±
1.8
>100
94.94 ±
1.9
>100
18.92 ±
9.8
19.70 ±
10.4
22.43 ±
16.6
22.15 ±
6.4
14.7 ± 22.2
>100
2.2. Biological evaluation
72.52 ±
14.6
34.78 ±
13.8
>100
>100
2.2.1. Antiproliferative activity of compounds 5(a–d) and 6(a–d)
The antiproliferative activity of compound 5(a–d) and 6(a–d) was
evaluated in four distinct types of human cancer cell lines (HeLa,
SKOV3, T47D and MCF-7). Normal human kidney cells (HEK293T) were
a
:The data are processed as the mean ± SD of three independent experiments
data.
Fig. 1. The synthesis route of compounds 6(a–d).
2

Z. Zhu et al.
Steroids 169 (2021) 108822
cholesteryl benzimidazole 6(a–d) efficiently yielded by esterification of
intermediate compound 5 with biotin. The antiproliferation effects of
compound 6 are summarized in Table 1. This biotinylated derivative
had a cell growth inhibition potency that was comparable to the lead
compound B-nor-cholesteryl benzimidazole. Of note, compound 6d
showed a better selectivity inhibition activity in SKOV3 cancer cells. As
compared to HEK293T, the compound 6d did not show any inactivation.
In conclusion, a biotin moiety was successfully introduced into the ste-
roidal structure without loss of the biological activity of the parental
compound. Thus, 6d was selected for further studies to identify a po-
tential chemotherapeutic agent.
were treated with compound 6d at different concentrations (0, 35, 40
and 45 μM) for 48 h, and the percentage of apoptotic cells was measured
by flow cytometry. Upon increasing the compound concentration, we
observed that most of the cells were apoptotic (19.94% versus 88.32%),
but the apoptotic state was mostly at the early stage (3.99% versus
59.37%). Thus, compound 6d exhibited an antiproliferative effect
possibly by inducing apoptosis in SKOV3 cells.
2.2.3. Transmemembrane mitochondrial analysis
DiOC6 (3) is a lipophilic fluorescent dye which used for labeling cell
membrane and hydrophobic tissue. It is an environment-sensitive type
of fluorescent dye whose fluorescence intensity is significantly enhanced
when it binds to membranes or to lipophilic biomolecules [7]. The re-
sults (Fig. 3) shown that the percentage of cells with depolarized mito-
chondria decreased from 99.9% to 52.9%, indicated that the ΔΨm
increased as compared to the control.
2.2.2. Cell apoptosis analysis
Apoptosis is the biological process underneath the programmed cell
death. Basically, there are two main forms of cell death: (i) necrosis,
which is also known as “pathological death”, and (ii) apoptosis, while
usually happens under certain physiological conditions [20]. Thus,
chemically induced apoptosis in tumor cells is pivotal in cancer therapy
[21]. FACS can be used to analyze the morphology and/or proliferative
status of a cell population. In this study, FACS was used to evaluate cell
fate (i.e. programmed cell death) upon treatment with different drug
concentrations. The four quadrants shown in Fig. 2 represent distinct
cell states: (i) dead cells (upper left); (ii) living cells (lower left); (iii) late
apoptotic cells (upper right), and (iv) early apoptotic cells (lower right)
[22]. To further certify that compound 6d induces apoptosis in SKOV3
cells, annexin V-FITC/PI staining was performed. For this, SKOV3 cells
2.2.4. Cell cycle analysis
In most of the eukaryotic cells, including tumor cells, the cell cycle is
divided in four stages: G1, S, G2, and M. According to each step of the
cell cycle, the following biological changes occur: (i) RNA and ribosomes
are mainly synthesized (G1 phase); (ii) enzymes needed for DNA repli-
cation are synthesized (S phase); (iii) large amounts of RNA and protein
are produced (G2 phase); and (iv) cell division effectively arise (M
phase) [23]. As a tool for cell cycle analysis, Propidium Iodide (PI) has
been extensively used due to its capacity to stain DNA. Since PI freely
Fig. 2. Compound 6d induce apoptosis in SKOV3 cells. Graphs indicate FACS analyses of cell populations treated with respective compound concentrations (0
(control), 35, 40 and 45 M) for 48 h.
μ
3

Z. Zhu et al.
Steroids 169 (2021) 108822
Fig. 3. The transmembrane potential of compound 6d (0 (control), 35 μM) induce on SKOV3 cells for 48 h.
penetrate broken cell walls, apoptotic nuclei can be stained. In contrast,
PI cann’t penetrate the walls of living cells. Therefore, PI-stained cells
were analyzed by flow cytometry, where both cell cycle and apoptotic
levels were evaluated [24]. Different concentrations of compound 6d (0,
indicates that 6d may blocks cell proliferation during the S phase.
2.2.5. Effect on apoptosis-related proteins
Disruption of the cell cycle regulation is one of the main reasons for
the abnormal proliferation of cancer cells. So, the modulation of the cell
cycle by certain therapeutic agents may lead to tumor growth arrest and,
ultimately, to apoptosis, therefore contributing to cancer therapy [25].
Cell cycle progression is cooperatively regulated by cyclin/cyclin-
dependent kinase (Cdk) complexes. Particularly, cyclin E/Cdk2 and
cyclin A/cdk2 complexes play key roles in the initiation and progression
35, 40 and 45 μM) were used to treat SKOV3 cells for 48 h, and then
stained with propidium iodide (PI), followed by flow cytometry analysis.
Non-treated cells were used as control. As shown in Fig. 4, control cells
at the G1, S and G2/M phases accounted for 96.11%, 3.30% and 0.58%,
respectively. Upon treatment with increasing amounts of compound 6d,
tumor cells at the S phase shifted from 3.30% to 31.90%. This result
Fig. 4. Cell cycle analysis of SKOV3 cells treated with increasing concentration of compound 6d (control), 35, 40 and 45 μM) for 48 h.
4

Z. Zhu et al.
Steroids 169 (2021) 108822
of the S phase [26]. In fact, S phase arrest of the cell cycle has been
associated with the activation of Cdk2-cyclin E/cyclin A and caspases in
ovarian cancer cells [27]. A Cdk inhibitor, p21, is known to be mediated
by cellular events such as cell cycle arrest, senescence and apoptosis
[28]. The functional activity of p21 is regulated by phosphorylation and
dephosphorylation of cdk proteins [29]. The p21 expression is modu-
lated by p53-dependent and independent mechanisms, and it is essential
for DNA damage-induced cell cycle arrest [30–32]. p21 can also sup-
press the expression of antiapoptotic genes, such as Bcl-2 and Bcl-xl
[33,34]. At the same time, Bax(a Bcl-2 family member) controls cell
death through activation of caspase-9 and caspase-3 [35]. Therefore,
using β-actin as a housekeeping/loading control, we further analyzed
the protein levels of upstream apoptosis-initiating protein (i.e. caspase-
9), downstream apoptosis-executing proteins (i.e. caspase-3), anti-and
pro-apoptotic proteins (Bcl-2 and Bax, respectively) and cyclin depen-
dent kinase inhibitors (i.e. p21), to determine whether SKOV3 cells
would be undergoing an apoptotic process.
Bcl-2. By comparing the mechanism of action of the lead compounds, it
can be found that both of them block the normal metabolism of tumor
cells by inhibiting the energy cycle in tumor cells. The proliferation
signal and transduction of tumor cells were inhibited, the protein syn-
thesis in tumor cells were slowed down, and the apoptosis of tumor cells
were induced. Overtly, compound 6d displays better activity in inhib-
iting SKOV3 cells through inducing apoptosis. Most interestingly, it has
low cytotoxicity to normal cells, which confers the potential for this
compound to be a therapeutic drug candidate for treatment of ovarian
cancer.
4. Experiment
4.1. Chemistry
Chemical reagents were purchased from J&K Scientific Co., Ltd.
(Shanghai, China) with the purity > 98%. Anhydrous reagents were
further purified by standard methods (when applicable). 1H NMR and
13C NMR spectra were recorded on a Bruker AV-300 NMR spectrometer
using indicated solvents (TMS as internal standard). In this case, the
values of the chemical shifts were expressed in δ values (ppm) and the
coupling constants (J) in Hz. Mass spectra and High-resolution mass
spectra (HR-MS) were carried out on Agilent LC QDECA-1000. The
melting points were determined on an X-4 binocular microscope melting
point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China) and
not modified. Infrared spectra were measured with a Thermo Scientific
Nicolet IS-10 Spectrophotometer using KBr pellets (Thermo Scientific,
America).
Thus, western blot analyses were performed, and the results are
summarized in Fig. 5. After 48 h of cell treatment, compound 6d was
able to significantly upregulate the expression of p21. Moreover, other
proteins including Bax, caspase-3, caspase-9, were also upregulated,
while the levels of anti-apoptotic Bcl-2 protein were suppressed. Taken
together, this data may indicate that 6d can induce cell apoptosis via the
mitochondria-dependent pathway.
3. Conclusions
In the present study, a series compounds with biotin group in the B-
nor-cholesterol benzimidazole structure were designed and synthesized.
The anti-proliferative activity of all targeted compounds was evaluated
by MTT. Among these compounds, the most effective was 6d, which
presented a selective cytotoxicity effect towards SKOV3 cell line, with
no effect on the control 293T cells. Therefore, the apoptotic properties of
compound 6d was further investigated by using the SKOV3 cell line.
Western blot analysis confirmed that 6d induces apoptosis by
mitochondrial-related pathways, whereas 6d increased the expression of
Bax, caspase-9, caspase-3 and p21, but decreased the relative levels of
4.1.1. General procedures to synthesize 2 ~ 4
Compound 1 (3.86 g, 10 mmol) was dissolved in 20 mL anhydrous
DCM with Et3N (3 mL, 15 mmol). Mixture was then dropped to ꢀ 15 ◦C
and stirred for 15 mins. MsCl (2 mL) was slowly added dropwise, and
reaction was continued for 5 mins. Compound mixture was concentrated
and purified by column chromatography (PE-EA 4:1elution.) to generate
compound 2.
Compound 2 (2.50 g, 5.38 mmol) was dissolved in 15 mL 1,4-
dioxane with glycol (3.41 g, 5.50 mmol). The reaction was kept at
90 ◦C for 18 h, then cooled to room temperature. The mixture was
extracted with EA. The combined layer was washed with water and
saturated NaCl solution sequentially, dried over anhydrous Na2SO4 and
filtered. The crude product was concentrated and purified by column
chromatography (PE-EA 2:1 elution) to generate compound 3.
Compound 3 (650 mg, 1.5 mmol) was dissolved in 20 mL DCM and 1
mL methanol. The reaction was maintained at ꢀ 80 ◦C for 15 mins, then
ventilated with ozone (2.5 mL/min) for 55 mins. Subsequently, dimethyl
sulfide (2.5 mL) was added and the mixture was keep stirring for 12 h at
room temperature. Compound mixture was concentrated and dissolved
in 25 mL methylbenzene, and 6 g Al2O3 was added and maintained in
reaction for 12 h at room temperature. Al2O3 was further filtered and
removed. The crude product was concentrated and purified by column
chromatography (PE-EA 1:1 elution) to generate compound 4.
4.1.2. General procedures to synthesize 5a-d and 6a-d
Compound 4 (463 mg, 1.0 mmol) and different kinds of o-phenyl-
enediamine (1.2 mmol) were respectively dissolved in 30 mL anhydrous
ethyl alcohol, and reaction proceeded for 8 h at 65 ◦C. The crude
products were concentrated and purified by column chromatography
(DCM-MeOH 15:1 elution) to generate compound 5(a–d).
Compound 5 (0.65 mmol), D-Biotin (711 mg, 2.9 mmol) and DCC
(801 mg, 3.8 mmol) were dissolved in 25 mL DCM with DMAP (119 mg,
0.97 mmol), and stirred for 12 h at 45 ◦C, respectively. The crude
products were concentrated and purified by column chromatography
(DCM-MeOH 10:1 elution) to generate compound 6(a–d).
Fig. 5. Western blot analysis of β-actin, p-21, Caspase 3, Caspase 9, Bcl-2 and
Baxproteins. SKOV3 cells were incubated with or without compound 6d, at
different concentrations, for 48 h. The levels of respective proteins were
detected using specific antibodies. Data shown are representative images from
three separate experiments.
5

Z. Zhu et al.
Steroids 169 (2021) 108822
4.2. Cell culture and reagents
incubation, each tub e with 1
μ
L DiOC6 (1
μ
M), blending and incubate
15 min in 37 ℃ incubator, the cells was centrifuged and discarded su-
HeLa (human cervical cancer), SKOV3 (human ovarian cancer),
T47D (breast cancer), MCF-7 (human breast cancer) and HEK293T
(normal renal epithelial) cells were cultured in DMEM medium sup-
plemented with 10% fetal bovine serum, 100 units/mL of penicillin, and
0.1 g/L streptomycin sulfate. Respective cell cultures were incubated at
37 ◦C in 5% CO2 incubator. All cell lines were purchased from Shanghai
Gaining biotechnology Co. Ltd (China) 0.3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT), and chemical reagents (pure
grade) were obtained from Sigma-Aldrich (St. Louis, MO). FITC-Annexin
V, PI and cell cycle kit was from BD (USA). Antibodies such as Bax, Bcl-2,
P-21, caspase-9, caspase-3, β-actin were from Cell Signaling Technology,
Inc (USA).
pernatant, then, each tube was cleaned with 1 mL warm PBS 2 times,
100 μL PBS was added into each tube, and the mitochondrial depolari-
zation was evaluated by BD AccuriTMC6 PLUS (USA).
4.7. Western blotting
SKOV3 cells were incubated with indicated doses of 6d (15, 30 and
45 μM) in triplicates for 48 h. Cells were then harvested and resuspended
in lysis buffer. Cell homogenates were centrifuged for 10 mins at 4 ◦C.
After determination of protein concentration, respective cell lysates
were resolved by sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (10% gel, SDS-PAGE) and transferred onto nitrocellulose
membranes. Membranes were blocked with 5% fat-free milk, and pro-
bed with respective primary antibodies, against Bcl-2, Bax, β-actin,
caspase-9, caspase-3 and p21, at 4 ◦C overnight. The bound antibodies
were detected using appropriate secondary antibodies and then visual-
ized with an enhanced chemiluminescent reagent [38].
4.3. MTT assay
The anti-proliferative activity of the synthesized compounds was
evaluated by MTT assay, in a 96-well format. Briefly, 100
μL of
respective cells, with a density of 104 cells/mL, were seeded into a 96-
well plate and incubated for 24 h at 37 ◦C in a humidified atmosphere
of 5% CO2. Thereafter, cells were treated with compounds, at different
Declaration of Competing Interest
concentrations. After 48 h, 20
μ
L MTT (5 mg/mL) was added to each
well and cells were incubated for more 4 h. Subsequently, 200
μ
L DMSO
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
was added to each well. The absorbance was measured on a Microplate
Reader (BIO-RAD) at the wavelength of 570 nm. The IC50 values were
calculated according to the inhibition ratios, (ODexperiment-ODblank)/
(ODcontrol-ODblank) × 100%. The data was processed by using GraphPad
Prism 8.0.2.
Acknowledgements
This research was supported by the National Natural Science Foun-
dation of China (21762008, 21562007), BAGUI Scholar Program of
Guangxi Province of China, Guangxi Natural Science Foundation of
China (2019JJA120066).
4.4. Cell apoptosis assay
SKOV3 cells were seeded in 6-well plates overnight, then different
concentrations of 6d were added for 48 h incubation (in triplicates).
After that, cells were washed with phosphate buffered saline (PBS)
twice, then resuspended in Annexin V binding buffer. Annexin V-FITC
was further added to the mixture, which was kept in dark for 15 mins. PI
was applied right before acquisition. After Annexin V and PI double
staining, apoptotic cells were analyzed, by flow cytometry (BD Calibur,
USA). For this, the percentage of cells positive for PI and/or Annexin V-
FITC was reported inside respective quadrants [36,37]. The percentage
of apoptotic cells was determined by flow cytometry, according to the
manufacturer’s instructions (BD Calibur, USA).
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