Introduction
Cancer, widely known as the "disease of the age" is a
complex genetic disorder defined as the conversion of a normal/healthy cell to a malignant one as a result
of various genetic changes such as mutations seen in
onco- and tumor suppressor genes, and developing
resistance to death, especially apoptosis.[,] The connection
between the induction of apoptotic cell death
and elimination of potential malignant cells, hyperplasia,
and tumor progression dates back to the early
1970s.[] Today, it is known that escape from apoptosis
is crucial in the transformation process to malignancy
[] and therefore the focus is on new approaches
targeting apoptosis regulation in cancer treatment.
[-] Apoptosis is the primary type of programmed
cell death induced when DNA damage is beyond repair.
The induction of apoptosis takes place through
two main pathways, namely the extrinsic and intrinsic
pathways.[] In the case of intrinsic pathway, various
stimuli directly affect their targets within the cell and a
series of events are initiated by the mitochondria. Basically,
all these stimuli cause the mitochondrial permeability
transition pores to open, a decrease in mitochondrial
transmembrane potential, and consequently,
the release of pro-apoptotic factors to the cytosol to
induce cellular death. The regulation of mitochondrial
pores is mainly governed by the Bcl-2 family proteins
consisting of a series of evolutionarily conserved proand
anti-apoptotic members that share Bcl-2 homology
(BH) domains. Among the pro-apoptotic members,
BH3-only proteins are the ones which share only
a small region of homology, the third Bcl-2 homology
(BH3) domain, with other Bcl-2 family members. The
so-called BH3-only proteins exert their pro-apoptotic
functions both by neutralizing the effects of other antiapoptotic
relatives and activating pro-apoptotic members
with multi-BH domains, which make them valuable
in breaking the resistance of cancer cells against
apoptotic death. Nevertheless, many types of tumors,
particularly those that are resistant to treatment, express
one or more of anti-apoptotic members at very
high levels and also carry mutations that inhibit the
induction of BH3-only proteins.[] On the other hand,
upon understanding the structures and interactions
of many Bcl-2 protein family members, it has become
possible to develop new generation BH3 mimetics that
may be alternative to traditional chemotherapy.[-]
In this study, we aimed to assess the cytotoxic
and apoptotic effects of clinically available navitoclax
(NTX) and venetoclax (VTX), which are members of
BH3 mimetics, and also their combination with apatinib
(APTB) on estrogen receptor (ER)-positive MCF-
7 and ER-negative MDA-MB-231 breast cancer cell
lines. Previous studies showed that both BH3 mimetics
have therapeutic properties on some hematological
malignancies, but various solid tumors develop resistance
to these drugs due to especially the increased expression
of Mcl-1, which is an anti-apoptotic member of the Bcl-2 protein family.[-] The APTB combination
with BH3 mimetics in the scope of present work
is a powerful inhibitor of the signal transduction pathway
dependent on vascular endothelial growth factor
(VEGF). VEGF is shown as a major angiogenic factor
in human cancers and is associated with failures in
treatment.[] As a matter of fact, it has been known
for a long time that serum and plasma VEGF levels of
metastatic breast cancer patients are also quite high.
[] Since VEGF is also known to up-regulate Mcl-
1 protein,[] it has been thought that the combined
application of the VEGF inhibitor APTB with the socalled
BH3 mimetics may offer a potential alternative
approach to traditional chemotherapy in breast cancer
treatment.
Methods
Cell Culture
The human breast adenocarcinoma cell lines (MCF-
7 and MDA-MB-231) were obtained from American
Type Culture Collection (ATCC, USA). Both cell lines
were cultured with high-glucose DMEM supplemented
with 100 IU/ml penicillin, 100 ?g/ml streptomycin
and 20% fetal bovine serum (FBS) in a humidified atmosphere
of 95% air with 5% CO2 at 37°C.
Cell Proliferation Assay
The effects of NTX and VTX, and also their combination
with APTB on the proliferation of breast cancer cell
lines were detected by MTT assay. Briefly, MCF-7 and
MDA-MB-231 cells were seeded into 96-well plates at a
density of 1x104 per well and allowed to attach for 24 h
before treatment. The cells were exposed to various concentrations
of NTX, VTX and APTB (0-100 µM) alone
or in combination for 24 h (The sole treatment of APTB
was carried out to determine non-toxic concentrations
of the inhibitor for further combined applications). After
incubation period, MTT solution (5 mg/ml PBS) was
added and the plates were located in an incubator with
5% CO2 at 37 ºC. After 4 h, MTT-formazan crystals were
dissolved in DMSO and then cell growth was assessed
by measuring the absorbance at 570 nm. Cell viability
was expressed as percentage survival, with 100% survival
taken as that observed in related control cells which
were treated with 0.1% and 0.2% DMSO for alone and
combined treatments, respectively.
Mcl-1 Protein Levels
Samples were assayed for Mcl-1 levels by using a commercially
available ELISA kit (RayBiotech, Inc., Norcross, GA, USA). Briefly, 100 µl of each standard or
sample were added to appropriate wells of the plate
pre-coated with an antibody specific for human Mcl-1.
The plate was incubated for 2.5 h at room temperature,
washed, and then all wells received 100 µl biotinylated
antibodies. Following 1 h incubation at room temperature,
plate was washed, 100 µl Streptavidin solutions
were added to each well and incubated for 45 min at
room temperature. After repetition of the washing step,
100 µl of TMB One-Step Substrate reagent was added
to each well for color development and incubated for
30 min at room temperature. The reaction was stopped
by adding 50 µl Stop solution to each well and the optical
density was measured at 450 nm. A Mcl-1 standard
curve was generated to quantitate the amount of Mcl-1
in ng/ml.
RNA Isolation and Quantitative Real Time PCR
(qRT-PCR)
RNA extraction from the samples was performed by
using RNeasy Plus Universal RNA Isolation Kit following
the manufacturer's protocol with slight modifications
(Qiagen, Redwood City, CA, USA). The RNA
concentration was determined using a NanoDrop
(MaestroNano micro-volume Spectrophotometer,
USA) and the RNA was reverse transcribed using a RevertAid
Reverse Transcriptase (ABM).[] cDNA was
stored at -80ºC for further use.
qRT-PCR analysis was carried out using SYBR
Green qPCR Master Mix (GM, Taiwan) in an Exicycler
96 Real Time Quantitative Thermal Block PCR System
(Bioneer, Daejeon, Korea). Beta-actin (ACTB) gene was chosen from the group of housekeeping genes to
normalize gene expression. The primer sequences for
the target genes and amplification conditions were given
in Table 1.
Table 1: Primer sequences and amplification conditions
Statistical Analysis
The data are presented as the mean±S.E.M of three
experiments. The differences in variance were analyzed
statistically using a one-way analysis of variance
(ANOVA) test by Graphpad prism 5.0 statistics software
(GraphPad, La Jolla, CA, USA). Tukey's test was
used as a post hoc.
Results
The Effects of BH3 Mimetics and Their Combinations
with Apatinib on the Proliferation of Human
Breast Cancer Cell Lines
Firstly, the effects of the BH3 mimetics and APTB
alone on the cell viability of human breast cancer cells
were investigated. For this purpose, MCF-7 and MDAMB-
231 cell lines were exposed to the drugs in the concentration
range of 0-100 ?M for 24 h and the results
were presented in Figure 1 and 2, respectively. As can
be seen from these figures, APTB was found non-toxic
for both cell lines at the concentrations of 2.5 and 5
µM and MDA-MB-231 cells were more sensitive than
MCF-7 cell line to higher concentrations of the inhibitor.
The results displayed that while the dose range of
NTX with increased cytotoxicity was determined as
0-50 and 0-5 µM on MCF-7 and MDA-MB-231 cells,
respectively, it was 0-50 µM for VTX on both cell lines.
Interestingly, more aggressive MDA-MB-231 cells
showed resistance to NTX at higher concentrations
and cell viability could not be reduced to below 48%
by increasing the mimetic concentration. Importantly,
it should be stated that relatively low concentrations of
both mimetics, 2.5 and 5 µM, could not reduce cell viability
of both two cell lines to less than 50% compared
to the their control groups.
To obtain more effective results in this relatively low
concentration range (0-5 µM), NTX and VTX were
combined with the non-toxic concentrations of APTB
(Figs. 3, 4). While MCF-7 cell proliferation could not be
decreased below 50% with these combined treatments,
it was acquired for MDA-MB-231 cell line with NTX
plus APTB applications. In the case of MCF-7, there
were no significant differences between the groups
with increased NTX plus constant APTB concentrations
or vice versa. On the other hand, approximately 58% cell viability was determined in 0.5 µM NTX plus
APTB treatments as with alone 5 µM NTX treatment
(Fig. 3a). These findings are significant in terms of getting
similar results with much lower concentration of
the mimetic. Unlike from NTX-APTB combined applications,
it was found that the most cytotoxic 5 µM
VTX plus 5 µM APTB treatment had also significantly
lower levels of cell viability than 5 µM VTX plus 2.5
?M APTB group (p<0.05) (Fig. 3b). When it comes to
MDA-MB-231 cell line, it was clearly seen that 2.5 µM
APTB combinations did not provide significant improvement
to the toxicity of VTX. However, when 2.5
µM VTX were combined with 5 µM APTB, cell viability
was significantly decreased from about 77% to about
63% (p<0.05). The effectiveness of the so-called combined
application was not able to significantly increase
by increasing VTX concentration to 5 µM (Fig. 4b). Finally,
Figure 4a shows that cell viability was about 45% in 0.5 µM NTX plus 5 µM APTB treatment and this
value could not be significantly decreased by increasing
NTX concentration as in the case of VTX. When all
above-mentioned results were evaluated together, the
apoptotic effects of 0.5 µM NTX plus 2.5 µM APTB, 0.5
µM NTX plus 5 µM APTB and 5 µM VTX plus 5 µM
APTB on MCF-7, and 0.5 µM NTX plus 5 µM APTB
and 2.5 µM VTX plus 5 µM APTB combinations on
MDA-MB-231 cell line were further investigated compared
to the sole applications.
Fig 1: The effects of APTB (a), NTX (b) and VTX (c) on cell proliferation of human breast cancer cell line MCF-7. The
cells were treated with 0.1% DMSO as control or three different agents at various concentrations for 24 h and cell
viability percentages were calculated compared with the control group. Data with error bars show the mean±S.E.M
of three experiments. **=p<0.01; ***=p<0.001; ****=p<0.0001 denote significant differences between control and
other studied groups by Tukey"s multiple range tests.
Fig 2: The effects of APTB (a), NTX (b) and VTX (c) on cell proliferation of human breast cancer cell line MDA-MB-231.
The cells were treated with 0.1% DMSO as control or three different agents at various concentrations for 24 h and cell
viability percentages were calculated compared with the control group. Data with error bars show the mean±S.E.M
of three experiments. ****=p<0.0001 denotes significant differences between control and other studied groups by
Tukey"s multiple range tests.
Fig 3: The effects of NTX (a) and VTX (b) plus APTB on
cell proliferation of human breast cancer cell line
MCF-7. The cells were treated with 0.2% DMSO
as control or NTX/VTX plus APTB combinations
at various concentrations for 24 h and cell
viability percentages were calculated compared
with the control group. Data with error bars show
the mean±S.E.M of three experiments. *=p<0.05;
**=p<0.01; ***=p<0.001; ****=p<0.0001 denote
significant differences between control and other
studied groups by Tukey"s multiple range tests.
Fig 4: The effects of NTX (a) and VTX (b) plus APTB
on cell proliferation of human breast cancer cell
line MDA-MB-231. The cells were treated with
0.2% DMSO as control or NTX/VTX plus APTB
combinations at various concentrations for 24
h and cell viability percentages were calculated
compared with the control group. Data with error
bars show the mean±S.E.M of three experiments.
**=p<0.01; ****=p<0.0001 denote significant
differences between control and other studied
groups by Tukey"s multiple range tests.
The Effects of BH3 Mimetics and Their Combinations
With Apatinib on the Mcl-1 Protein Levels of
Human Breast Cancer Cell Lines
As stated above, similar or better results in cytotoxicity
assessment with much lower concentrations of BH3
mimetics could be obtained by combining these mimetics
with non-toxic dosages of APTB. To enlighten
the role of Mcl-1 protein levels in these synergic cytotoxic
effects of the so-called combinations, the changes
in these protein levels were determined with ELISA
kit and compared with alone treatments. As is known,
Mcl-1 is one of the anti-apoptotic members of the Bcl-2
protein family which regulates mitochondrial pore formation
and among the factors that are held responsible
for the resistance of solid tumors to BH3 mimetics.[] The results were presented in Figure 5. Among comparable
groups, the only significant difference was found
between control and 5 µM VTX and between 2.5 and
5 µM APTB groups of MCF-7 cells (p<0.05). Unfortunately,
significant decreases in Mcl-1 protein levels
could not be attained with the application of combined
treatments in both cell lines.
Fig 5: The effects of alone APTB, alone BH3 mimetic and
combined BH3 mimetic plus APTB treatments on
Mcl-1 protein levels of human breast cancer cell
lines MCF-7 (a) and MDA-MB-231 (b). The cells
were treated with 0.1 or 0.2% DMSO as control
groups or agents at various concentrations for 24
h and Mcl-1 protein levels were calculated. Data
with error bars show the mean±S.E.M of three
experiments. *=p<0.05 denotes significant differences
between 0.1% DMSO control and other
studied group by Tukey"s multiple range tests.
The Effects of BH3 Mimetics and Their Combinations
With Apatinib on Pro- and Anti-Apoptotic
Gene Regulation of Human Breast Cancer Cell Lines
Transcriptional regulation of pro-apoptotic caspase-3
(Cas3), Bak and Bax, and anti-apoptotic Bcl-2, Bcl-xL
and Mcl-1 genes in two different human breast cancer
cell lines which were exposed to BH3 mimetic and
APTB alone or in combination at the concentrations
determined as mentioned above were investigated. As
very well known, all studied genes except Cas3 are the
members of the Bcl-2 gene family. The effector protein
Cas3 can be considered as the intersection and the last
step of different apoptotic cell death cascades. The effects
of the agents and their combinations on the transcriptional
regulation of the specified genes in MCF-7
and MDA-MB-231 cell lines are presented in Tables 2
and 3, respectively. From the obtained results, it was
seen that to combine 0.5 µM NTX with 2.5 µM APTB caused significant induction in the expression of proapoptotic
adaptor Bak and Bax proteins when compared
with both control group and alone 0.5 µM NTX
and 2.5 µM APTB applications (p<0.0001) in MCF-7
cell line. However, this combined treatment also significantly
induced anti-apoptotic Bcl-2 and Mcl-1 expression
compared with control. When 0.5 µM NTX
or 2.5 µM APTB was applied alone, either there was
no significant change or significantly lower induction
by comparison with the so-called combined treatment
(p<0.0001) were observed in the expression levels of
these anti-apoptotic genes, respectively. On the other hand, when we consider the interactions between proand
anti-apoptotic relatives of Bcl-2 protein family,[]
this situation can be interpreted as the response against
increased Bax and Bak protein expression. Surprisingly,
to increase APTB concentration from 2.5 to 5 µM in
the combined application also gave rise to induction in
the expression of Bcl-2 and Mcl-1 proteins without any
induction in pro-apoptotic ones. Differently from NTX
plus APTB combination, 5 µM APTB combination of 5
µM VTX could significantly increase Cas3 expression
besides significant induction in the expression of Bak
and Bax. As indicated in Table 2, although higher induction
in Bak and Bax expression were observed in 5
µM VTX when compared to combined treatment, Cas3
expression did not significantly increase. So, it would
not be wrong to say that VTX and APTB combination
showed synergistic effect in terms of apoptotic death
of human breast cancer cell MCF-7. Unfortunately, the
highest induction in the anti-apoptotic gene expression
was also found for this combination, which can be
commented as a parameter reducing the effectiveness
of this dual drug administration. Contrary to the promising
results seen on MCF-7, no significant increases in
pro-apoptotic gene expression have been observed in
combined applications performed on MDA-MB-231
cell line (Table 3). Conversely, the sharpest increase in
Bcl-2 gene expression and significant down regulation
in Cas3 were determined in this cell line that treated
with 5 µM APTB + 0.5 µM NTX. The significant down
regulation of Mcl-1 gene in the same combined group
also could not be seen as an improved resistance to survival
due to the similar results that obtained both in
the alone 5 µM APTB and 0.5 µM NTX treated groups.
Although, significant up-regulation of Bcl-xL gene in
0.5 µM NTX group was repressed by the combination
with 5 µM APTB, it is clear that higher sensitivity of more aggressive MDA-MB-231 cells to the combined
tested agents rather than MCF-7 is independent from
the factors investigated.
Table 2: The expression level of the selected genes in MCF-7 cell line
Table 3: The expression level of the selected genes in MDA-MB-231 cell line
Discussion
The Bcl-2 protein family controls the mitochondrial
pathway of apoptotic cell death depend on the levels
and interactions of its pro- and anti-apoptotic members.
On the other hand, the findings obtained until
now show us that several anti-apoptotic Bcl-2 family
proteins, such as Bcl-2, Bcl-xL and Mcl-1 are expressed
at high levels in many cancer types including breast
adenocarcinoma.[-] Hence, it is crucial to specifically
target these anti-apoptotic proteins in terms of
developing novel and effective treatments with fewer
side effects. In this context, the mimetics of BH3-only
proteins are thought to be powerful agents against cancer.[] NTX, which can target Bcl-2/Bcl-xL, and Bcl-
2 specific VTX are among the BH3 mimetics which
were proved to be active in lymphoid malignancies and
studies which are being conducted on for other types
of tumors.[-] Of course, it should be remembered
that the impression of any BH3 mimetic can extend
beyond its targeted protein.[-] Nevertheless, other
non-targeted anti-apoptotic members, such as Mcl-
1, are held responsible for the resistance of especially
solid tumors to these drugs and focus is on combined
studies.
In the current study, we analyzed the cytotoxic
and apoptotic effects of NTX/VTX and their combination
with APTB on two different breast cancer cell
lines with different aggressiveness for the first time. We
observed that 0?5 ?M APTB alone was non-toxic on
both MCF-7 and MDA-MB-231 cell lines and the latter
one was more sensitive to the agent in its higher doses
within 24 h. In a recent study of Gao et al., supportive cytotoxicity results were shown on MDA-MB-231
cells for 72 h.[] It was determined that APTB exerted
dose-dependent inhibition of MCF-7 cell growth at
the concentrations higher than 2 µM after 48 h treatment,
which is also compatible with our data.[] According
to the literature, APTB shows its cytotoxic effect
on cancer cells by blocking the phosphorylation
of VEGFR-2 and thereby restraining several signaling
pathways.[] On the other hand, as stated previously,
it was found that there has been correlation between
VEGF and Mcl-1 expressions on certain hematological
malignancies,[,] which give rise to thought that
BH3 mimetics plus APTB can show synergistic effects
in terms of inhibition of solid tumor cell proliferation.
As a matter of fact, we found that to combine NTX/
VTX with non-toxic concentrations of APTB significantly
reduced the mimetic concentrations required to
achieve similar or higher cytotoxic effects when compared
to their sole applications. In other words, APTB
combination sensitized the cells against the so-called
BH3 mimetics in correlation with their aggressiveness.
Tutusaus et al., indicated the synergistic effect of NTX
plus sorafenib, a multikinase inhibitor, on hepatocellular
carcinoma depend on the changes in the Bcl-2
protein profile.[] In another recent study, tyrosine
kinase inhibitors were shown to drive prostate cancer
apoptosis when they were combined with Bcl-xL/
Bcl-2 inhibitors by increasing Mcl-1 degradation.[]
However, we observed significant up-regulation in
Bcl-2 expression in both MCF-7 and MDA-MB-231
cell lines, which were exposed to the combined treatments.
Mcl-1 expression was also significantly up-regulated
in MCF-7 cells. For these reasons, we could not
explain the synergistic cytotoxic effects of NTX/VTX
plus APTB over the changes in Bcl-2 and Mcl-1 gene
expression against human breast cancer cells. After all,
the more sensitivity of aggressive MDA-MB-231 cells to the so-called combined applications and significant
Bak/Bax and Cas3 expression in less aggressive MCF-
7 cells can be seen as encouraging results. So, we can
suggest that VTX/NTX plus APTB in their indicated
low concentrations might be used efficaciously on human
breast cancer cells but further studies are needed
to clearly elucidate the observed synergistic cytotoxic
effects.
Conclusion
Our results suggested that NTX/VTX and APTB
showed cytotoxic effects on breast cancer cell lines
depending on cell aggressiveness. Additionally, in this
study, it was demonstrated that APTB sensitized breast
cancer cells to low doses of NTX/VTX again in correlation
with the aggressivity. Hopefully, these combinations
can provide new options for novel approaches to
the treatment of breast cancer. However, the underlying
mechanism(s) in these synergistic cytotoxic effects
should be elicited with further studies.
Limitations of the Study
Within the scope of this study, expression levels of a
limited number of genes could be examined. In order
to elucidate the exact mechanisms leading to the cytotoxic
effects of the studied applications, more target
behaviors should be investigated.
Acknowledgments: This study was supported by Scientific
Research Projects Unit of Pamukkale University (PAUBAP2019BSP008).
Peer-review: Externally peer-reviewed.
Conflict of Interest: None declared.
Financial Support: None declared.
Authorship contributions: Concept - B.K.Y., Ö.Ö.A.,
A.S., A.Ş.; Design - B.K.Y., Ö.Ö.A., A.S., A.Ş.; Supervision
- B.K.Y., Ö.Ö.A., A.S., A.Ş.; Funding - B.K.Y., Ö.Ö.A., A.S.,
A.Ş.; Materials - B.K.Y., Ö.Ö.A., A.S., A.Ş.; Data collection &/
or processing - B.K.Y., Ö.Ö.A.; Analysis and/or interpretation
- B.K.Y., Ö.Ö.A., A.S.; Literature search - B.K.Y., Ö.Ö.A.;
Writing - B.K.Y., Ö.Ö.A., A.S.; Critical review - B.K.Y.,
Ö.Ö.A., A.S., A.Ş.
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