Effects of Sorafenib and Arsenic Trioxide on U937 and KG-1
Cell Lines: Apoptosis or Autophagy?
The first two authors equally contributed to this work.
Haghi A, Salami M, Mohammadi Kian M, Nikbakht M, Mohammadi S, Chahardouli B, Rostami Sh, Malekzadeh K. Effects of sorafenib and arsenic
trioxide on U937 and KG-1 cell lines: apoptosis or autophagy? Cell J. 2020; 22(3): 253-262. doi: 10.22074/cellj.2020.6728.
Acute myeloid leukemia (AML) is a clonal disorder of hemopoietic progenitor cells. The Raf serine/threonine (Ser/Thr) protein kinase isoforms including B-Raf and RAF1, are the upstream in the MAPK cascade that play essential functions in regulating cellular proliferation and survival. Activated autophagy-related genes have a dual role in both cell death and cell survival in cancer cells. The cytotoxic activities of arsenic trioxide (ATO) were widely assessed in many cancers. Sorafenib is known as a multikinase inhibitor which acts through suppression of Ser/Thr kinase Raf that was reported to have a key role in tumor cell signaling, proliferation, and angiogenesis. In this study, we examined the combination effect of ATO and sorafenib in AML cell lines.
Materials and Methods
In this experimental study, we studied
Data demonstrated that sorafenib, ATO, and their combination significantly increase the number of apoptotic
cells. We found that the combination of ATO and sorafenib significantly reduces the viability of U937 and KG-1 cells.
The expression level of selective autophagy genes,
The expression levels of apoptotic and autophagy activator genes were increased in response to treatment. The crosstalk between apoptosis and autophagy is a complicated mechanism and further investigations seem to be necessary.
Acute myeloid leukemia (AML) as a malignant disease of the bone marrow, is caused by acquired somatic mutations and chromosomal rearrangements which occur in a hematopoietic progenitor. Regardless of its etiology, the AML pathogenesis involves extraordinary differentiation and proliferation of a clonal population of myeloid stem cells. Different processes involved in leukemia are controlled by signaling pathways initiated by activated receptor tyrosine kinases (RTKs) (1).
RAS is a downstream factor for various RTKs. Activation of RAS signaling pathway has a critical function in the development of human malignancies (2). Fundamental activity of the RAS pathways arises from downstream effectors of RAS, activating mutations in the RAS, or even overexpression of a variety of RTKs, including vascular endothelial growth factor receptors (VEGFRs), epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor (PDGFR) (3). Therefore, RAS mutations or activation in human tumors could lead to cell survival and proliferation. RAS adjusts multiple pathways such as RAF/MEK/ERK pathway which remarkably activate cellular transformation
RAF kinases are serine/threonine protein kinases which act as a downstream effector of RAS. The Raf serine/ threonine protein kinase isoforms including A-Raf, B-Raf and Raf1, are the upstream in the MAPK cascade (4) and they regulate cellular proliferation and survival. Moreover, it was recently demonstrated that wild-type Raf1 could, independently of MAPK signaling, promote cell survival, through interactions with apoptosis and antiapoptosis regulatory proteins (5).
Beclin1 (which is encoded by
Sorafenib is known as a multikinase inhibitor which
has effective roles in tumor cell signaling, proliferation,
and angiogenesis (Fig .1A,) (10). Arsenic trioxide (ATO)
targets various cellular functions through multiple
molecular factors (Fig .1B,). ATO plays dual roles in
acute promyelocytic leukemia (APL) cells, and at low
concentrations, it activates differentiation while at high
concentrations, it promotes apoptosis (11). The aim of the
present study was to appraise the combination effect of
ATO and sorafenib on
Materials and Methods
The antiproliferative activity of ATO (0.5-5 μM) and sorafenib (2-12 μM) was assessed using MTT assay at 24, 48 and 72 hours, to distinguish optimal conditions with maximum effects, in KG-1 and U937 cells. In order to determine the growth inhibitory effects of ATO and sorafenib, KG-1 and U937 cells were seeded into 96- well plates at a primary density of 5×103 per well (100 µl). After that, cells were treated with ATO, sorafenib and their combinations for 24, 48 and 72 hours. Control cells were treated with 0.1% DMSO alone. The proliferation rate of cells was analyzed by MTT assay and results are expressed as proliferation rate.
Cell lines and treatment
We purchased U937 and KG-1 cell lines from the National Cell Bank of Iran (Pasteur Institute, Iran). Cell lines were cultured and expanded in RPMI 1640 supplemented with 10 and 20% heat-inactivated FBS for U937 and KG-1 cell line, respectively, 100 IU/ ml penicillin and 100 μg/ml streptomycin. Cells were cultured in a CO2 incubator at 37˚C with 5% CO2 in a humidified atmosphere. Cells were seeded at 1×105 cells/ mL. For treatment experiments, prior to each assay, 80- 90% confluent flask was centrifuged, the supernatant was discarded and each cell pellet was resuspended separately in 1-2 ml of media and completely pipetted to prevent cell clumping. Then, 10 μL of cell solution including cell and media, was pipetted and cells were counted. Afterward, the cells were treated with the selected concentrations.
To assess the percentage of apoptosis induced by the above-noted compounds, fluorescein-conjugated annexin-V (annexin-V-FITC) staining assay was accomplished based on the manufacturer’s protocol. We treated KG-1 and U937 cells with ATO (1.618 and 2 μM for KG-1 and 1 μM for U937) and sorafenib (7 μM for KG-1 and 5 μM for U937) and their combination for 48 hours. Data acquisition and analysis of apoptosis by a Becton Dickinson (BD, America) flow cytometer and percentage of the annexin-V+/PI- cells was recorded; finally, we used flowJo program to analyze our data.
Cell cycle analysis
Here, U937 and KG-1 cell population were treated with specific concentrations of ATO and sorafenib for 48 hours, then fixed in cold 70% ethanol and stained with propidium iodide (PI). Cells were evaluated by BD flow cytometer instrument and data were analyzed by flowJo program. The apoptotic cell fraction was calculated based on hypodiploid G0/G1 DNA fraction.
RNA isolation and real-time polymerase chain reaction
We treated KG-1 and U937 cells with ATO (1.618 and
2 μM for KG-1 and 1 μM for U937) and sorafenib (7 μM
for KG-1 and 5 μM for U937) and their combination for
48 hours. Treated cells were harvested and dissolved in
1 ml of TRI pure (Roche Applied Science, Germany),
based on the manufacturer’s instructions. DEPCtreated water was used to reconstitute the RNA pellets.
The quantity and quality of total RNA were analyzed
spectrophotometrically using Nanodrop ND-1000
(Nanodrop Technologies, Wilmington, DE) at 260 and
280 nm. Then, complementary DNAs (cDNAs) were
reverse transcribed from 1-2 µg of total RNA by use of a
cDNA synthesis kit (Takara Bio Inc., Japan) according
to the manufacturer’s instructions. The concentration
of cDNA was normalized in series of PCR through
Data were analyzed using GraphPad Prism 5 software by using one/two way ANOVA and for post-test evaluations, we used t test. All data represent the results obtained from triplicate independent experiments and expressed as mean ± standard errors of the mean (SE). Asterisks (*, **, and ***) in the Figures indicate P<0.05, P<0.01, and P<0.001, respectively.
|Gene||Primer sequence (5ˊ-3ˊ)||Reference|
|F: CAG CCC TGT CCA GTA GC||(21)|
|R: GCG TGA CTT TAC TGT TGC|
Evaluation of cell proliferation using MTT test
Metabolic activity can be detected through measuring the activity of succinate dehydrogenase as a mitochondrial enzyme via MTT assay. We applied the MTT assay to determine the anti-proliferative activity of ATO and sorafenib (alone and in combination) in U937 and KG-1 cell lines.
We perceived both time- and dose-dependent effect of compounds. As seen in Figure 2, we did not see a significant difference between 48 and 72 hours treatment as assessed by two way ANOVA. Our data indicated that combination effect of ATO and sorafenib (P<0.001 for both cell lines) compared to the control or even singlecompound treatment (P<0.001 for KG-1 and P<0.01 for U937), could significantly decrease cell proliferation at 48 hours in both U937 and KG-1 cell lines (Fig .2,).
To investigate apoptosis and necrosis, we performed flow cytometry assay using annexin-V FITC/PI staining for both U937 and KG-1 cell lines following 48h treatment. As seen in Figures 3A and B,, our result indicated an increase in apoptotic cells (annexin+/PI) and minimum percentage of necrosis in treated cells compared to control, in both U937 and KG-1 cells. Moreover, we observed a significant increase (up to 70% in KG-1 and around 80% in U937 cells) in combination doses (P<0.001). The percentages of apoptotic cells in treated KG-1 and U937 cell lines were significantly higher than those of the control groups.
Cell cycle assay
DNA content of U937 and KG-1 cells was assessed by flow cytometry. To specify the apoptosis activating role of ATO and sorafenib, U937 and KG-1 cells were treated with chosen doses for 48 hours. Our result indicated that combination of ATO and sorafenib increased hypodiploid G0/G1 DNA fraction in a dose-dependent manner (1.13 to 8.3% for KG-1 cell and 9.21 to 16.1% for U937 cell) (Fig .4,).
Real-time polymerase chain reaction assay
In order to investigate the mechanisms underlying the
synergy observed for ATO and sorafenib, we analyzed
gene expression of
U937 cells were treated with specific concentrations of
ATO (1 μM), sorafenib (5 μM) and their combination for
48 hours. We observed that the expression of
KG-1 cells were treated with ATO (1.618 and 2 μM),
sorafenib (7 μM) and their combination for 48 hours. Our
data indicated that the expression of
In the present research, we tried to assess the
ATO as a multi-target agent is able to activate apoptosis and autophagy (27) through various molecular pathways in numerous cancers including solid tumor cells and hematological malignancies. In this study, we observed ATO cytotoxic and apoptosis-inducing effects in both U937 and KG-1 cell lines in a dose and time-dependent manner. Our data indicated that ATO can influence cell proliferation and cell death pathway. We examined a wide range of ATO concentrations in both resistant and sensitive cell lines. We observed that 1.618 and 2 μM of ATO has a significant effect as compared to its lower concentrations in KG-1 (as a resistant cell line). Chiu et al. (28) reported that ATO in combination with ionizing radiation may enhance programmed cell death by activating both autophagy and apoptosis in human fibrosarcoma cells. Also, Chiu et al. (29) confirmed that ATO can synergistically activate both apoptosis and autophagy.
Sorafenib is known as a multikinase inhibitor which act
through suppression of Ser/Thr kinase Raf that is known
to have an important role in tumor cell signaling and
proliferation, and various RTKs involved in angiogenesis,
such as VEGF (30). However, sorafenib was shown to be
more effective in leukemia with the FLT3-ITD mutation,
and its antileukemic function was clarified in several
patients with AML and wild-type form of FLT3 (31).
In our previous study, we demonstrated that sorafenib
downregulates the gene expression of
The RAF/MEK/ERK signaling pathway was shown to
be activated in various processes in cancer. In the present
study, we observed that the expression of B-RAF, MEK1,
MEK2, and RAF1 increased as a result of treatment
with ATO, sorafenib and their combination in KG-1 cell
line. In addition, the expression level of
We observed that the expression of
Tai et al. (38) reported that sorafenib-induced
autophagy signaling pathway through significant
induction of LC3-II in HCC cell lines. Shimizu et al. (39)
demonstrated increased expression of
In this study, we found that combination of ATO and sorafenib significantly reduced the viability of U937 and KG-1 cells. In addition, the crosstalk between apoptosis and autophagy is complicated and varies among different cell types. Similar stimuli may activate both pathways as they share various signaling. ATO with antileukemic activity in AML cell lines, enhances the antitumor activity of sorafenib in both U937 and KG-1 cells. Our study indicated a potential mechanism underlying the interaction between ATO and sorafenib in U937 and KG-1 cell lines.
This study had financial support by the Hematology, Oncology and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences. The authors declare no conflicts of interest.
A.H., M.N., S.M.; Contributed to conception and design. A.H., M.S., M.M.K., B.C., S.R., K.M.; Contributed to all experimental work, data and statistical analysis, and interpretation of data. M.N., S.M.; Were responsible for overall supervision. A.H.; Drafted the manuscript. All authors read and approved the final manuscript.