Cancer Biomarkers 31 (2021) 13–25 13

DOI 10.3233/CBM-203257
IOS Press

Salidroside induces cell apoptosis and inhibits

the invasiveness of HT29 colorectal cells by
regulating protein kinase R, NF-κB and
STAT3
Attalla F. El-kotta,b,∗ , Eman R. ElBealyc , Ali S. Alshehria , Ayman E. El-Kenawyd ,

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Heba S. Khalifab and Amira M. AlRamlawye
a
Biology Department, College of Science, King Khalid University, Abha, Saudi Arabia

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b
Zoology Department, College of Science, Damanhour University, Damanhour, Egypt
c
Biology Department, College of Science for Girls, King Khalid University, Abha, Saudi Arabia
d
Pathology Department, College of Medicine, Taif University, Saudi Arabia
e
Mansoura Research Centre for Cord Stem Cell (MARC-CSC), Stem Cells Bank, Children’s Hospital, Mansoura
University, Mansoura, Egypt
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Received 28 December 2020
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Accepted 28 February 2021

Abstract.
BACKGROUND: Protein kinase R (PKR) can suppress various types of solid tumors by inducing cellular oxidative stress and
apoptosis. Likewise, Slaidorside, a plant flavonoid, was shown to have anti-tumorigenesis in many solid tumors.
OBJECTIVE: This study evaluated anti-tumorigenesis of Salidroside in HT29 colorectal cancer and investigated if the underlying
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mechanism involves activation of PKR.

METHODS: Control or PKR deficient cells were cultured in DMEM media treated with 100 µM Salidroside and cell survival,
apoptosis, and other biochemical-related markers were evaluated.
RESULTS: Salidroside significantly reduced cell survival and proliferation and increased the release of lactate dehydrogenase
(LDH) and levels of single-stranded DNA (ssDNA). It also increased the protein levels of caspases 3 and 8. Concomitantly,
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Salidroside increased the protein level and activity of PKR and increased the expression of its downstream targets, p-eIF2α (Ser51 ),
p53 MAPK, and p53. On the contrary, it inhibited the nuclear activation of STAT-3 and NF-κB p65. In PKR deficient cells, the
partial effects of Salidroside on cell survival, proliferation, and apoptotic markers were observed coincided with no effects on the
expression of eIF-2α, and JNK, p53, p38 MAPK, and caspase 8 but with a significant decrease in the nuclear activities of STAT3
and NF-κB.
CONCLUSION: Salidroside suppresses the tumorigenesis of HT29 CRC by increasing activation of eIF-2α and JNK and
upregulation of p53, p38 MAPK, and caspase-8 through upregulating and activation of PKR. However, the tumor suppressor effect
of Salidroside requires also inhibition of STAT3 and NF-κB in a PKR-independent mechanism.

Keywords: Salidroside, colorectal, HT29, protein kinase R, STAT3, eIF-2α, NF-κB

1. Introduction

∗ Corresponding
author: Attalla F. El-kott, Biology Department,
College of Science, King Khalid University, Abha, Saudi Arabia. Colorectal cancer (CRC) is one of the most lethal
E-mail: elkottaf@kku.edu.sa; elkottaf@sci.dmu.edu.eg. cancers in old and young males and females worldwide

ISSN 1574-0153/$35.00 c 2021 – IOS Press. All rights reserved.

14 A.F. El-kott et al. / Salidroside effects against HT29 colorectal cells
(> 30% in both sexes) with a projection to increase
by 60% in 2030 [1]. Risk factors for CRC include a
high meat diet and smoking [1,2]. Reactive oxygen
species (ROS) and inflammation remain the major well-
known tumorigenic pathways in CRC [2,3]. Unfortu-
nately, CRC still a very complicated disorder that re-
quires more exploration and specific drug development.
Currently, several lines of evidence have pointed out
the emerging role of the protein kinase R (PKR) in the
pathogenesis of numerous types of solid tumors and
other chronic inflammatory disorders and metabolic dis-
orders, as well as, neurodegenerative disorders [4–6]. In
general, PKR is a 551 amino acid long serine-threonine
kinase that is encoded in humans by the EIF2AK2
gene [4,6]. It is mainly induced by double-stranded
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RNA (dsRNA) and interferon-γ (INF-γ) during the vi-
ral pathogen invasion and plays a significant role as an
antiviral agent through suppression of mRNA transcrip-
tion, protein synthesis, and viral replication, as well as
induction of cell apoptosis [5,7,8]. However, the up-
regulation and activation of PKR can also be induced
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in other non-viral mechanisms, including inflamma-
tory cytokines, energy excess, over-nutrition, lipid ac-
cumulation, calcium stress, irradiation, reactive oxy-
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gen species and oxidative stress, endoplasmic reticu-
lum (ER stress), Lipo-stress, amyloid-β (Aβ) peptide
accumulation, and many drugs [4,5].
However, the contribution of PKR in tumorigenesis
of different types of solid tumors was shown to be a
dual effect of either promoting or suppressing tumor
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progression. Besides, PKR levels and activity were sig-
nificantly increased or decreased in Serval types of tu-
mors [5,9]. Indeed, reduced levels and activation of
PKR were shown in head, skin, lung, and colon cancer
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where its activation inhibited tumor progression and
improved the prognosis, thus suggesting a tumor sup-
pressor effect of this enzyme [10–13]. On the other
hand, another line of evidence has shown overexpres-
sion of PKR in thyroid, colon, breast, liver, lung, and
skin cancers, as well as in acute myeloid leukaemia,
thus challenging the above-mentioned studies that PKR
is a tumor suppressor [9,14–18]. Such duality of ef-
fects was shown due to different experimental settings
(in vivo vs in vitro) and tumor type and stage and is
dependent on other activated signaling pathways, thus
leaving a debate on the overall effect of PKR function
in cancer [5].
However, the tumor suppressor effect of PKR is be-
lieved to be due to its ability to induce cell apopto-
sis and arresting the cell cycle through its wider effect
on various signalling pathways that control these path-
ways. Within this view, it was shown that PKR induces
cell apoptosis by phosphorylation and activation and
of the Eukaryotic Initiation Factor 2 (eIF-2α), thus im-
pairing its activity which ultimately leads to inhibition
of protein synthesis [4,5]. In addition, PKR can induce
cell apoptosis and inhibit cancer cell invasiveness in
an EIF-2α-independent manner and through different
mechanisms, including increasing the transcription and
translation of Fas and apoptotic Bcl-2 effector proteins,
activation of caspases 3/8/9 [19,20]. Furthermore, PKR
induces cell apoptosis through interaction with P53 and
inhibits cell invasiveness through upregulation of ATF-
3 transcription factor [5,9]. Besides, PKR is a major
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stimulator of the apoptotic signalling pathways, includ-
ing kinase (JNK) and p38 mitogen-activated protein
kinase [21,22]. On the contrary, PKR has been shown
to activate the transcription factor nuclear factor-kappa
beta (NF-κB) which plays a significant role in promot-
ing tumor proliferation, growth, stemness, and inva-
sion [5,19].
Nonetheless, the plant Kingdome remains a rich
source of anticancer-derived drugs, some of which
having minimal side effects rather than synthetic
drugs [23]. Salidroside is a major flavonoids glycoside
isolated from Rhodiola rosea (R. rosea L) [24]. Cov-
ering data are showing a potent tumor-suppressive ef-
fect of Slaidorside against a variety of solid tumors,
including thyroid, renal, bladder, breast, lung, CRC,
and colon cancers [25–27]. Among all these studies,
the confirmed mechanisms of protection involved inhi-
bition of ROS generation, expression of BCl2 and sig-
nalling through PI3K/Akt/mTOR, as well as activation
of caspase-3 dependent cell apoptosis, and inhibition of
JAK2/STAT3.
However, the effect of Salidroside on the expression
and activation of PKR was never investigated either
in normal or cancer cells. Therefore, in this study, we
aimed to investigate if the tumor suppressor effect of
Salidroside is mediated, at least, by modulating the
expression, activation, and downstream signalling of
PKR in the HT29 CRC cell line. Also, the expression
of serval PRK downstream targets including JNK, P38
MAPK, NF-κB, ATF-3, STAT1/STAT3 were targeted.
2. Materials and methods
2.1. Cell culture and treatments
The Human CRC cell line, HT29 was purchased from
the American Type Culture (USA) and were cultured (at
 A.F. El-kott et al. / Salidroside effects against HT29 colorectal cells
< 85% confluent) in DMEM supplied with 10% heat-
inactivated fetal bovine serum (FBS), 1% penicillin,
and 0.1% streptomycin (ThermoFisher Scientific) (5%
CO2, 37◦ C) (He et al., 2017). They were seeded in and
incubated under similar conditions for 24 h.
2.2. Small interfering RNAs (siRNAs) and cell
treatment
PKR siRNA (h) (Cat. No. sc-36263), Control siR-
NAs (Cat. No. sc-37007), siRNA Transfection Reagent
(Cat. No. sc-29528), siRNA Transfection Medium (Cat.
No. sc-36868) and siRNA Dilution Buffer (Cat. No.
sc-29527) were purchased from Santa Cruz Biotech-
nology, TX, USA. Transfection reagent was performed
according to the manufacturer’s instructions. In brief,
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cells were initially seeded at a density of seed 2 × 105
cells per well in a 2 ml antibiotic-free normal growth
medium supplemented with FB in O2 incubator at 37◦ C
in a CO2 incubator to reach a confluence of 60–80%.
The siRNA of PKR (6 µl of 10 µM) or control siRNA
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(8 µl of 10 µM) as well as the transfection reagent
(6 µl) were diluted individually with 100 µl transfec-
tion media and then mixed and incubated for 30 min to
prepare the transfection reagent working mixture. The
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transfection working mixture was added to 0.8 ml of the
transfection media (free of serum and antibiotic) and
the whole 1 ml mixture was overlaid onto the washed
cells and incubated for 5 h. after that, 1 ml of 2x growth
media was added to each transfection and incubated
for 18 h which was then removed and replaced with
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1x growth media and further incubated for 48 h. To
confirmed the results, protein expression of PKR was
confirmed by western blotting.
2.3. Cell treatment
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Depending on the experimental procedures, cell
(HT29 or knocked-down HT29 cells) were cultured at
a density of 2 × 104 (5% CO2 , 37◦ C) in 96 well plates
in the growth culture media and treated with Salidro-
side (Cat. No. SMB00072, Sigma Aldrich, St Louis,
MO, USA) at a final concentration of 100 µM. This
concentration was based on the initial dose-response
curve of the effect of increasing doses (0–200 µM)
of Salidroside on cell survival, proliferation, levels of
lactate dehydrogenase (LDH), and cell apoptosis.
2.4. Determination of cell viability
The rate of cell viability was measured using a cell
viability counting kit-8 (CCK-8) (Cat No. CK04-13,
Dojindo, Japan) in accordance with the manufacturer’s
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instruction. In brief, 100 µl of the growth media was dis-
carded and replaced with a new fresh medium contain-
ing 10% of working CCK8 solution, incubated for 3 h
at 37◦ C and reading the absorbance (ABS) at 450 nm
(Spectramax plate reader; Model M2, Molecular De-
vices, USA).
2.5. Measurements of LDH activity
All treated cells were centrifuged (200xg, 5 min,
4◦ C) to collect the supernatants. LDH activity in all
collected supernatant was measured using a commercial
kit (Cat. No. ab102526; Abcam, Cambridge, UK). In
brief, the test relies on the conversion of NAD+ in the
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presence of LDH to NADPH which can be measured
and detected using a special probe. In the test, 2 µl LDH
substrate buffer was mixed with 48 µl of LDH assay
buffer to generate a reaction mixture that was added to
each sample or standard (20 µl). The measurements of
the ABS were performed at different time intervals at
450 nm (Spectramax plate reader; Model M2, Molecu-
lar Devices, USA) and LDH levels were obtained from
the standard curve.
2.6. Measurement of cell proliferation
Cell proliferation was determined by Bromo-20 -
deoxyuridine (BrdU) ALISA kit (Cat. No. 11647229-
001; Roche Diagnostics, Indianapolis, IN, USA). The
principle of the test relies on the incorporation of BrdU
into newly synthesized cellular DNA, which can be then
detected by using specific BrdU-peroxidase (POD) and
a substrate reagent, 3, 30 , 5, 50 -tetramethylbenzidine.
Briefly, the cells were cultured with various treatments
in a final volume of 100 µl for 24 h. then10 µl BrdU
was added to each well and incubated for 2 h at 37◦ C.
Then, the media were removed, and 200 µl FixDena
was added to each well and incubated for 30 min at
23◦ C. The FixDena solution was removed and 100 µl
POD solution was added to each well and incubated
at 23◦ C for 90 min. The solution was then removed
and each well was washed three times with 200 µl of
the washing solution (PBS). Then 10 µl of substrate
reagent was added to each well and incubated at 23◦ C
for 15 min until the color was developed. Absorbance
was read at 370 nm with a reference range at 492 nm
(Spectramax plate reader, model M2, Molecular De-
vices, USA). Cell proliferation was calculated as the
percent of controls.
2.7. Determination of apoptosis (ssDNA assay)
The levels of ssDNA in all tested samples were mea-
16 A.F. El-kott et al. / Salidroside effects against HT29 colorectal cells
sured using an ELISA kit (Cat No. APT225, Millipore,
USA) per the manufacturer’s instruction. Briefly, the
cells were centrifuged (200xg, 5 min, 4◦ C) to collect
pellets. Pellets were then fixed with 200 µl of the sup-
plied fixative reagent and kept at 23◦ C for 30 min, after
which the fixative was discarded and all plates were
dried at 23◦ C for 2 h. After that, 100 µl of formamide
solution was added to each well, incubated the plate
was incubated at 23◦ C for 10 min, and then heated at
75◦ C for 10 min to denature DNA. Then, all plates
were cooled at 4◦ C and the formamide was removed.
Next, 3% 100 µl skim milk (prepared in distilled water)
was added to all wells and incubated for 1 h at 37◦ C.
Then, the milk was removed and cells of all wells were
incubated with 100 µl mouse monoclonal anti-ssDNA
antibody (supplied with the kit) for 30 min at 23◦ C,
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then washed with 250 µl of 1x of the provided washing
buffer (3 times). Then, 200 µl of the provided HRP-
secondary anti-mouse secondary antibody was added
incubated for 30 min at 23◦ C. After that, 100 µl ABTS
solution was added to all wells and incubated for an-
other for 30 min at 23◦ C. to allow sufficient binding
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of the HRP. The reaction was terminated by the addi-
tion of 100 µl stop solution and the ABS was read at
405 nm (Spectramax plate reader, Model M2, molecular
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devices, USA).
2.8. Migration and invasion assays
The migration and invasion rates of both cell lines
were, according to the method previously described in
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our laboratories [28]. For cell migration, the cells (5 ×
104 cells, 100 µl) were plated on the top of the Tran-
swell chambers (8-µm pores and a membrane diameter
of 6.5 mm) (Corning Costar, NY, USA) and treated as
mentioned whereas the lower chamber was filled with
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500 µl 10%. The whole setting was incubated for 24 h.
Then, the cells in the upper chamber were wiped and
those in the lower chamber were fixed with methanol
for 10 min at 23◦ C and then stained with 0.2% crystal
violet for another 10 min at 25◦ C. Stained cells were
counted using a light microscope in 4 random fields and
average readings were presented. Cell invasion was per-
formed in the same way, but using Corning Transwell
chambers of 5-mm pore size. Before the test, the upper
chamber was first coated with 50 µl Matrigel (Cat. No.
DLW354263, Sigma Aldrich, UK). During the invasion
test, cells were first starved in a serum-free medium for
3 h and then loaded onto the upper chamber.
2.9. Western blots
Cell pellets of all treatments were homogenized in
250 µl RIPA buffer lysis kit (Cat. No. 89900, Ther-
moFisher) supplied with 5 µl protease inhibitor cock-
tail (Cat. No. P8340 Sigma-Aldrich, MO, USA). The
samples were centrifuged 10000xg for 10 min to col-
lect the supernatant which was collected and stored
at −80◦ C until use. Protein levels in all fractions
were determined by a Bradford assay using a com-
mercial kit (Cat. No 23300, ThemoFisher Scientific,
MA, USA). The Western blot procedure was per-
formed as previously described in our labs [29]. Pro-
teins from all groups (40 µg/well) were separated by
SDS Page and transferred onto nitrocellulose mem-
branes. Membranes were first incubated, for 2h at room
temperature, with rotation, with the primary antibod-
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ies against PKR (Cat. No. 3072, 1:1000, 74 kDa), p-
PKR (Thr451 ) (Cat. No. 3075, 1:1000, 74 kDa), eIF-
2α (Cat. No. 5324, 1:1000, 38 kDa), p-eIF-2α (Ser51 )
(Cat. No. 5324, 1:1000, 38 kDa), JNK (Cat. No. 9252,
1:1000, 46/54 kDa), p-JNK (Thr183 /Tyr185 ) (Cat. No.
9255, 1:1000, 46/54 kDa), p38 MAPK (Cat. No. 8690,
1:1000, 40 kDa), p53 (Cat. No. 2527, 1:1000, 53 kDa),
STAT3 (Cat. No. 9139, 1:1000, 86 kDa), p-STAT3
(Tyr705 ) (Cat. No. 9145, 1:1000, 53 kDa), NF-kβ p65
(Cat. No. 8242, 1:1000, 65kDa), p-NF-κB p65 (Ser356 )
(Cat. No. 3033, 1:1000, 53 kDa), cleaved caspase-3
(Cat. No. 9661, 17/19 kDa, 1:500), caspaspe-8 (Cat. No.
9746, 1:1000, 57 kDa), β-actin (Cat. No. 3700, 45 kDa,
1:2000), lamin B1 (Cat. No. 12255, 1:1000, 68 kDa)
(Cell signalling Technology, MA, USA). Next, mem-
branes were incubated with appropriate corresponding
horseradish peroxidase (HRP)-conjugated secondary
antibody for 2 h at room temperature with rotation.
All antibodies were prepared in TBST buffer and all
membranes. Membranes were stripped with a commer-
cially available stripping buffer (Cat. No. 21059, Ther-
moFisher Scientific) up to 5 times and the detection of
the phosphorylated forms was done first, and the β-actin
the last. A control known sample was run on all gels
for normalization of the target protein. Band intensities
were detected using a Pierce ECL kit (ThermoFisher,
USA, Piscataway, NJ), scanned using a C-Di Git blot
scanner (LI-COR, NE, USA), and analyzed using the
scanner associated software.
2.10. Statistical analysis
GraphPad Prism statistical software package (V8)
was used for the analysis of data. Data were analyzed
by one-way ANOVA, followed by Duncan’s Multiple
Range Test. The significance among parameters was
determined at p < 0.05. Data are presented as mean ±
SD.
 A.F. El-kott et al. / Salidroside effects against HT29 colorectal cells 17

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Fig. 1. Salidroside induces a dose-response, decrease in cell survival (A) and cell proliferation (B), and a dose-response increase in LDH releases
(C) and content of single-stranded DNA (ssDNA) (D) in HT29 colorectal cell line. HT29 cells were cultured in DMEM for 24 h under 5% CO2
and 37◦ C and treated with increasing concentrations of Salidroside (0–200 µM). Data are presented as mean ± SD of three experiments/treatment
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each performed in triplicate. a: vs. control (0.0 µM), b: vs. 10 µM, c: vs. 20 µM, d: vs. 40 µM, e: vs. 60 µM, and f: vs. 80 µM.

3. Results and B). These data confirm the tumor suppressor ef-
fect of Salidroside and highlight the involvement of
3.1. Salidroside inhibits HT29 survival and activation of PKR/eIF-2α in this process.
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invasiveness and stimulates cell death, at least by
activation of PKR/eIF-2α
To test the tumor suppressor effect of Salidroside on
the HT29 CRC cell line, we have treated the cells with
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On the other hand, the deletion of PKR significantly
reduced the eIF-2α levels (Fig. 2B), partially reduced
cell survival and proliferation (Fig. 3A and B), and par-
tially increased levels of LDH and ssDNA (Fig. 3C and
D), as compared to control cells. Also, it significantly

increasing concentrations of the drug. Salidroside, in

a dose-dependent manner, decreased cell survival and
proliferation and increased the release of LDH and lev-
els of ssDNA, a marker of cell apoptosis (Fig. 1A–D).
The maximum effect of Salidroside on these parameters
was shown at concentrations of 100, 150, and 200 µM
where no significant variation was seen between them
(Fig. 1A–D). When tested at a dose of 100 µM, Salidro-
side significantly increased the protein levels of PKR,
p-PKR (Thr451 ), and p-eIF-2α (Ser51 ) as compared
to control cells, thus indicating upregulation of PKR
and activation of both PKR and eIF-2α (Fig. 2A and
B). Concomitantly, Salidroside at a concentration of
100 µM significantly reduced cell survival and prolifer-
ation (Fig. 3A and B), increased LDH release and ss-
DNA levels (Fig. 3C and D), and suppressed cell migra-
tion and invasion as compared to control cell (Fig. 4A
increased cell migration and invasion as compared to
control cells (Fig. 4A and B), thus indicating that basal
levels of PKR are crucial for tumorigenesis of HT29
cells.
However, when Salidroside was incubated with PKR-
deficient HT29 cells, it failed to alter the expression of
eIF-2α, partially decreased cell survival and prolifera-
tion, partially increased levels of LDH and ssDNA, and
partially suppressed cell migration and invasion as com-
pared to control + Salidroside-treated cells (Fig. 2B,
Fig. 3A–D, and Fig. 4A and B). Although basal lev-
els of PKR are shown to promote CRC progression in
HT29 cells, these data suggest that activation of PKR
by Salidroside is a tumor suppressor event but not the
only signaling pathway induced but Salidroside.
18 A.F. El-kott et al. / Salidroside effects against HT29 colorectal cells

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Fig. 2. Salidroside increases protein level and activity of protein kinase R (PKR) and subsequently activates the Eukaryotic Initiation Factor 2
(eIF-2α) in HT29 colorectal cells. HT29 control or PKR deficient cells were cultured in DMEM for 24 h under 5% CO2 and 37◦ C and treated
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with Salidroside (100 µM). Data are presented as mean ± SD of three experiments/treatment each performed in triplicate. a: vs. control (untreated
control cells) (Lane 1), b: vs. Salidroside-treated control cells (Lane 2), c: vs. PKR-deficient cells (Lane 3). Lane 4 represents a sample that was
taken from PKR-deficient cells + Salidroside.
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3.2. Salidroside activates JNK and upregulated p53 3.3. Salidroside upregulates caspase-8 only in control
and p38 MAPK only in control cells, but inhibits cells but upregulates caspase-3 in both control
NF-κB and STAT3 in both control and and PKR-deficient cells
PKR-deficient cells
Treating the HT29 cells with Salidroside significantly
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JNK, p38, p53, NK-κB, and STAT-3 are major targets
of PKR (Garcia et al., 2017). Indeed, with no alteration
in total levels of JNK and nuclear levels of NF-κB and
STAT3 deletion of PKR in HT29 cells significantly re-
duced the total protein levels of p-JNK and nuclear lev-
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upregulated caspase-3 and caspase-8 as compared to
control cells (Fig. 7A and B). However, the deletion of
PKR did not affect the protein levels of caspase-8 but
partially reduced protein levels of caspase-3 as com-
pared to control cells (Fig. 7A and B). Notably, treating

els of p-NF-κB (Ser358 ) and p-STAT3 (Ty705 ) (Fig. 5A–
C) with a parallel decrease in total protein levels of p38
MAPK and p53 (Fig. 6A and B). On the other hand,
treating the HT29 cells with 100 µM Salidroside for
24 h significantly increased levels of p-JNK, p53, p38
MAPK but significantly lowered nuclear levels of p-
NF-κB (Ser358 ) and p-STAT3 (Ty705 ) (Fig. 5A–C and
Fig. 6A and B). Interestingly, although Salidroside was
unable to alter protein levels of p-JNK, p53, and p38
MAPK in PKR-deficient cells as compared to PKR-
deficient cells, levels of p-NF-κB (Ser358 ) and p-STAT3
(Ty705 ) showed the maximum decrease in the PKR-
deficient cells + Salidroside (Fig. 5A–C and Fig. 6A
and B). These data suggest that Salidroside induced ac-
tivation of JNK, p38, and MAPK are PKR-dependent.
Besides, it can be concluded that Salidroside is a potent
inhibitor of NF-κB and STAT3.
the PKR deficient cells with Salidroside didn’t affect
protein levels, but further reduced the expression of
caspase-8 as compared to PKR-deficient cells (Fig. 7A
and B). These data suggest that the upregulation of
caspase-8 by Salidroside is PKR-dependent, whereas
the upregulation of caspase-3 involves at least PKR but
requires activation of other pathways.
4. Discussion
In this study, we have confirmed the tumor suppres-
sor effect of Salidroside in an HT29 CRC cell line and
have shown that it is not only able to induce cell apop-
tosis and inhibit proliferation but also suppress their in-
vasion and migration. However, the novelist finding of
this study is that we are showing a collaborative mecha-
 A.F. El-kott et al. / Salidroside effects against HT29 colorectal cells 19

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Fig. 3. Deletion of protein kinase R (PKR) partially ameliorates the inhibitory effect of Salidroside on cell survival (A) and proliferation (B) and its
stimulatory effect on LDH release (C) and the increase in levels of single-stranded DNA (ssDNA) (D) in HT29 colorectal cell line. HT29 control
or PKR deficient cells were cultured in DMEM for 24 h under 5% CO2 and 37◦ C and treated with Salidroside (100 µM). Data are presented as
mean ± SD of three experiments/treatment each performed in triplicate. a: vs. control (untreated control cells), b: vs. Salidroside-treated control
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cells, c: vs. PKR-deficient cells.

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Fig. 4. Deletion of PKR increases cell invasion (A) and migration (B) whereas treatment of Salidroside inhibits these events in both control and
protein kinase R-deficient HT29 colorectal cells. HT29 control or PKR deficient cells were cultured in DMEM for 24 h under 5% CO2 and 37◦ C
and treated with Salidroside (100 µM). Data are presented as mean ± SD of three experiments/treatment each performed in triplicate. a: vs. control
(untreated control cells), b: vs. Salidroside-treated control cells, c: vs. PKR-deficient cells.
20 A.F. El-kott et al. / Salidroside effects against HT29 colorectal cells

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Fig. 5. Salidroside increases the activation of JNK only in control HT29 colorectal cells, but inhibits the nuclear activity of NF-κB and STAT3 in
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both control and protein kinase R (PKR) -deficient cells. HT29 control or PKR-deficient cells were cultured in DMEM for 24 h under 5% CO2 and
37◦ C and treated with Salidroside (100 µM). Data are presented as mean ± SD of three experiments/treatment each performed in triplicate. a: vs.
control (untreated control cells) (Lane 1), b: vs. Salidroside-treated control cells (Lane 2), c: vs. PKR-deficient cells (Lane 3). Lane 4 represents a
sample that was taken from PKR-deficient cells + Salidroside.

nism by which Salidroside exerts its anti-tumorigenesis
effects which involve 1) upregulation and activation of
PKR and subsequent upregulation of p38 MAPK and
p53 and activation of eIF-2α and JNK activation and 2)
PKR independent mechanisms which involve inhibition
of NF-κB and STAT3 activation.
Initially, Salidroside, and in a dose-dependent man-
ner inhibited HT29 cell proliferation and survival and
significantly stimulated their death. These data were
also confirmed by a sustained increase in cleaved
caspase-3, which is considered a classic marker of cell
death. Such findings remain confirmatory to many other
previous studies that have shown a tumor suppressor
effect of Salidroside against many solid tumors includ-
ing the CRC. Indeed, Salidroside inhibited cell growth,
proliferation, and invasion of MG63 and U2OS hu-
man osteosarcoma, WRO differentiated thyroid, and
SW1116 colon cancer cell lines through inhibition of
the JAK2/STAT-3 signalling pathway [27,30,31]. Also,
Salidroside inhibited the growth, proliferation, and in-
vasion of A549 lung cancer cells and suppressed metas-
tasis in breast cancer cells by inhibiting ROS gener-
ation and activation of p38 MAPK [32]. In HCT-116
CRC cancer cells, Salidroside inhibited cell survival
 A.F. El-kott et al. / Salidroside effects against HT29 colorectal cells 21

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Fig. 6. Salidroside upregulated p38 MAPK and p53 only in control HT29 colorectal cells. HT29 control or PKR-deficient cells were cultured in
DMEM for 24 h under 5% CO2 and 37◦ C and treated with Salidroside (100 µM). Data are presented as mean ± SD of three experiments/treatment
each performed in triplicate. a: vs. control (untreated control cells) (Lane 1), b: vs. Salidroside-treated control cells (Lane 2), c: vs. PKR-deficient
cells (Lane 3). Lane 4 represents a sample that was taken from PKR-deficient cells + Salidroside.
C
OR
TH
AU

Fig. 7. Salidroside downregulates casapase8 only in control HT29 colorectal cells, but downregulates caspase-3 in both control protein kinase R
(PKR)-deficient cells. HT29 control or PKR-deficient cells were cultured in DMEM for 24 h under 5% CO2 and 37◦ C and treated with Salidroside
(100 µM). Data are presented as mean ± SD of three experiments/treatment each performed in triplicate. a: vs. control (untreated control cells), b:
vs. Salidroside-treated control cells, c: vs. PKR-deficient cells.

and induced cell death by inhibiting different targets, in-
cluding mTOR, STAT3, and NF-κB. However, in HT29
CRC, Salidroside tumor-suppressive effect was medi-
ated by inhibition of the PI3K/Akt/mTOR signaling
axis [33].
On the other hand, a unique finding in this study is
that we also report a new protein target of Salidroside
in HT29 CRC. Herein, we are providing the first evi-
dence in the literature that treatment with Salidroside
may affect cell function by inducing an upregulation
and activation of PKR. We have specifically targeted
this protein due to its multifunction on different intra-
cellular cell signalling and its pre-established role in
cancer initiation and progression [6,8]. In general, PKR
is an interferon (IFN) -inducible double-stranded RNA
(dsRNA) protein kinase that normally responds to viral
infection by inhibiting transcription, translation, and
proliferation, thus inducing cell apoptosis [6]. How-
22 A.F. El-kott et al. / Salidroside effects against HT29 colorectal cells
ever, the activation of PKR requires several conforma-
tional changes in which homodimerization was shown
to be mandatory for PKR activation. Also, autophos-
phorylation at sites Thr446 and Thr451 are indispensable
for PKR homodimerization and increased its catalytic
activity [7,8]. This has been also shown in this study
where Salidroside didn’t only increase the protein lev-
els of PKR but also increased its phosphorylation at
Thr451 , thus activation.
Under normal conditions, the cytoplasmic levels of
PKR in normal cells are kept at low levels [6]. How-
ever, under a viral infection or other stress conditions
such as ROS, UV radiation, ER stress, inflammatory
cytokines, etc., cytoplasmic levels and activity of PKR
are rapidly increased [5,6]. In cancer cells, levels of
OP
PKR were either increased or decreased and had contra-
dictory effects of either promoting or inhibiting cell sur-
vival [5,9]. Indeed, reduced levels of PKR were shown
on the neck, head, melanoma, lung, and colon cancers
and were positively correlated with the prognosis of
the disease [10,13]. As an apoptotic kinase, PKR can
C
induce cell apoptosis by phosphorylation and activa-
tion of eIF-2α (at Ser51 ) and by direct activation of the
FADD/caspase-8/caspase- 3 and caspase-9/APAF path-
OR
ways [13,20]. However, the tumorigenic role of PKR is
mediated by activation of STAT-3 and NK-κB [4,5].
Associated with the increase in the level and activity
of PKR and cell death in Salidroside-treated control
HT29 cells, we have also found a significant increase
in phosphorylation of eIF-2α at Ser51 and higher pro-
TH
tein levels of caspase-3 and caspase-8 in these cells.
These data suggest a possible role of PKR in regulating
the eIF-2α pathway and acting as a tumor suppressor.
To confirm this, we have silenced PKR in these cells
AU
and then treated them with the highest effective dose
of Salidroside. Interestingly, this resulted in partial ef-
fects of Salidroside on cell survival, LDH release, and
cell death. Also, Salidroside failed to increase levels
of elF-2α in PKR-deficient cells, partially increased
protein levels of caspase-3, and partially inhibited cell
migration and invasion. These data suggest that even in
the absence of PKR, Salidroside was still able to induce
cell death and inhibit cell survival but at a lower level.
Based on these data, it becomes clear to us that the anti-
proliferative, anti-invasiveness, and apoptotic effect of
Salidroside in H29 cells involves multiple mechanisms
in which activation of the PKR/eIF-2α pathway is just
one mechanism and other signaling pathways are in-
volved. Besides, it suggests that CRC in HT29 cells is
a very complicated process and involves the activation
of PKR and other signalling pathways. Furthermore,
these data suggest that the activation of caspase-8 by
Salidroside is completely PKR-dependent.
Interestingly, the deletion of PKR alone partially
reduced cell survival and cell proliferation and par-
tially increased LDH release, cell death, and levels of
caspases-3. It also and completely prevented the in-
crease in caspase-8 and phosphorylation of eIF-2α as
compared to control cells. Another important finding is
that deletion of PKR increased cell migration and in-
vasion, even one could expect that the deletion of PKR
will increase cell proliferation and inhibit cell apoptosis
(by assuming to act as a tumor suppressor through de-
creasing eIF-2α), these data suggest that the basal levels
Y
of PKR in HT29 CRC cells may also play a significant
role in HT29 maintaining tumor progression. Such par-
tial tumorigenic effects observed in PKR-deficient cells
could be explained by the concomitant reduction in the
activation of STAT-3 and NF-κB (as discussed later)
which normally promote cancer progression and inhibit
tumor apoptosis. However, our data still validate that
sustained activation of PKR by Salidroside stimulates
partial cell apoptosis thus suggesting a PKR to act as
a tumor suppressor effect after Salidroside treatment
mainly through increasing the activation of eIF-2α. It
should be also mentioned here that the phosphorylation
of eIF-2α is under the control of other kinases rather
than PKR including (PKR) -like endoplasmic reticu-
lum kinase (PERK); general control non-derepressible
2 kinase (GCN2), and heme-regulated eIF2a kinase
(HRI) [4,5]. However, since Salidroside was unable to
phosphorylate eIF2a in PKR deficient cells, these data
suggest that PKR is the most targeted kinase on which
Salidroside acts to inhibit eIF-2α.
Nonetheless, PKR could also induce cell apoptosis
in other mechanisms that are completely independent
of eIF-2α including activation of p53, JNK, and p38
MAPK [5]. p53 induces cytochrome C release and ac-
tivates mitochondria-mediated (intrinsic) cell death by
upregulation of the Bcl-2-like protein 4 (Bax) [34].
However, JNK can result in cell apoptosis by increasing
the production of jBID that enhances the Smac release
from the mitochondria to induce activation of caspase-
8 [35]. Besides, both JNK and p38 MAPK induced cell
apoptosis by activation of Bcl-2 pro-apoptotic mem-
bers such as Bim [36]. Additional extra mechanisms
by which JNK and p53 acts as inducers of caspase-
dependent cell death are well-explained somewhere
else [37,38].
In this study, Salidroside also increased JNK ac-
tivation and increased protein levels of p53 and p38
MAPK. However, Salidroside was unable to stimulate
 A.F. El-kott et al. / Salidroside effects against HT29 colorectal cells
these apoptotic markers in PKR deficient HT29 cells,
thus indicating that Saldiorside-induced activation of
p38 MAPK, p53, and JNK is PKR-dependent. Given
that caspase-8 is mainly induced by JNK, such a PKR-
dependent increase in JNK could explain why the up-
regulation of caspase-8 was also PKR dependent. Al-
though the effect of Salidroside on MAPK activation
was poorly investigated in tumor cell lines, the pro-
tective effect of Salidroside on breast and lung cancer
cells was mediated by inhibition of p38MAPK which
contradicts our findings. Such variation could be re-
lated to variation in cell lines, treatment, doses used,
and experimental conditions.
On the other hand, cancer is described as an in-
flammatory disorder where inflammation creates a suit-
OP
able environment needed for tumor growth, metasta-
sis, and stemness [39]. NF-κB and STAT-3 are two re-
lated inflammatory transcription factors that play ac-
tive roles in promoting cancer development, progres-
sion, invasion, and metastasis of most solid tumors [40].
It was shown that a cross-talk exists between STAT3
C
and NF-κB where inflammatory cytokines (i.e. IL-6)
induce sustained activation of NF-κB and STAT3, in
turn, promotes the activation of NF-κB [41–43]. In this
OR
regard, the tumor suppressor effect of NF-κB is me-
diated by the expression of numerous anti-apoptotic
genes such as the members of the B-cell lymphoma-2
(Bcl-2) family, the caspase-8 inhibitor, FLIP, and the
inhibitor of apoptosis proteins including c-IAP1/2 and
XIAP [40]. Similarly, STAT-3 can stimulate cycle pro-
TH
gression by stimulating the expression of CyclnD1, c-
Myc and Survivin and inhibit cell apoptosis by upregu-
lating numerous anti-apoptotic protein such as Bcl-2,
B-cell lymphoma-2-like 1 (Bcl-XL) and myeloid cell
AU
leukaemia sequence (Mcl1) [44–47]. In addition, NF-
κB induced angiogenesis and stimulates cancer metas-
tasis by increasing the expression of vascular endothe-
lial growth factor (VEGF) and matrix metallopeptidase
(MMPs) [40]. Similarly, STAT-3 induces MMPs and
other metastatic mediators such as high-mobility group
box 1 (HMGB1), Twist, Vimentin, and Snail [43].
Nevertheless, the activities of NF-κB and STAT3
were significantly increased in the colon and CRC [48].
It was shown that that PKR can stimulate the activity
of both NF-κB and STAT-3 in cancer cells [5]. Indeed,
the deletion of PKR in HT29 cells of this study in-
hibited the nuclear activity of both STAT3 and NF-κB
which support its regulatory effect on these parame-
ters. As mentioned previously, this could explain why
PKR deficient cells showed a partial reduction in cell
survival and proliferation and partial activation of cell
23
apoptosis. Hence, given its stimulatory effect on PKR,
it is reasonable to think that Salidroside may activate
these markers to challenge its anti-apoptotic effect. Un-
expectedly, we found significantly lower activities of
STAT3 and NF-κB in Salidroside-treated control cells.
Besides, there was a maximum decrease in the activities
of STAT-3 and NF-κB in PKR deficient cells co-treated
with Salidroside. Based on these data, and in addition to
stimulation of PKR/eIF-2α/P53/JNK/p38 MAPK path-
way, it becomes obvious that inhibition of NF-κB and
STAT-3 are other mechanisms by which Salidroside
induces CRC cell death. This could explain why there
was a partial reduction in cell survival and prolifera-
Y
tion and partial induction of LDH release, apoptosis,
and caspaspe3/8 activation in Salidroside-treated PKR
deficient-cells as compared to Salidroside-treated con-
trol cells. Supporting our data, the inhibitory role of
Salidroside on NF-κB and STAT3 was shown in normal
and cancer cells, including thyroid cancer, osteosar-
coma, and CRC [27,30,31,48,49]. Although not investi-
gated here, a recent study has shown that the inhibitory
role of Salidroside on NF-κB and STAT3 involves acti-
vation of AMPK and SIRT1 [50].
In conclusion, our study is the first to show that the
Salidroside tumor suppressor effect in HT29 CRC cells
requires concomitant activation of PKR and inhibition
of STAT3 and NF-κB. These data are very encouraging
for future clinical trials.
Acknowledgments
The authors extend their appreciation to the dean-
ship of Scientific Research at King Khalid University,
Abha, KSA for supporting this work under grant num-
ber (R.G.P.2/80/41). Also, this work was supported
by the Taif University Researchers Supporting Project
Number (TURSP-2020/99), Taif University, Taif, Saudi
Arabia.
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