Abstract

Hepatocellular carcinoma is one of the most debilitating cancers with increased level of
resistance to chemotherapy. Increasing evidence demonstrated that combination therapy
can help eradicate HCC. Berberine is thought to enhance antitumor activity of different
chemotherapeutic drugs, though its effect on combination with regorafenib in HCC is
unclear. This study was performed to explore the effect of this combination on HepG2 cells.
Combined treatment caused significant inhibition of proliferation and increased apoptosis
compared to each drug alone, it also led to reduction of IC50 of regorafenib by 1.46-fold, and
berberine by 4.2-fold while in combination. Effect of combination on different pathways was
analysed through measuring protein levels of major regulators of these pathways. Protein
levels of p-Akt, p-ERK and NF-κB p65 was measured by ELISA revealing synergistic
reduction in combined group compared to each drug alone. Combination also showed
significant reduction in VEGF protein level, thus tumor angiogenesis. CyclinD1 protein level
was analysed by ELISA to reveal synergistic reduction in combination treated group. Utilizing
flow cytometry technique, Annexin V/ PI staining was used and showed increased apoptosis
in combination group compared to each drug alone. Effect of combination on autophagy was
also measured through Beclin1 protein level by western blot and found synergistic reduction
compared to each alone. Our results infer that combining regorafenib and berberine lead to
synergistic interaction and dose reduction of each of them, enhanced cell cycle regression
and apoptosis. Combination also affected different signalling pathways involved in various
oncogenic processes. Inhibited angiogenesis, and reduced autophagy.
Introduction
Globally, primary liver cancer is the sixth most diagnosed cancer per year and of cancer
related deaths it comes the third[1]. Hepatocellular carcinoma (HCC) is the cancer of
hepatocytes and accounts for 75%-85% of primary liver cancer[1]. Different types of
mutations occur in HCC tumor cells that dysregulate various regulatory pathways and switch
it to act upon cancer pernicious benefits[2]. Along with overlapping and cross talking that
takes place between these pathways that render understanding hepatocarcinogenesis more
complicated. Though, much research had outlined the most altered pathways in HCC
involved into many crucial cell processes such as differentiation, proliferation, cell survival
and angiogenesis[3][4].

Receptor tyrosine kinase pathways plays pivotal role in HCC carcinogenesis. Growth factors
such as VEGF, HGF, IGF-1, IGF-2, PDGF, TGFβ, EGF and FGF when bind to its
corresponding tyrosine kinase receptors, activate multitudinous downstream signals in
different pathways including PI3K/AKT/mTOR, Ras/MAPK signalling pathways[5]. Upon
activation of these pathways, its downstream members can regulate cytosol targets and
translocate to nucleus to phosphorylate an array of transcription factors that regulate
expression of genes involved in protein synthesis, metabolism, and crucial to cell survival,
proliferation and angiogenesis[6] [4].

PI3K/AKT/mTOR is a critical pathway involved in initiation and progression of many cancers

including HCC. It is found to be hyperactivated in 50% of HCC patients with more aggressive
tumor [7], where p-Akt overexpression having the strongest association with poorer
prognosis[8].

MAPK pathways is frequently dysregulated in HCC, MEK1/2 phosphorylation is increased

seven times in tumor tissues contrasted to adjacent intact hepatic tissue[4][9]. MAPK
pathway inhibitors such as Ribosomal S6 Kinase 2 (RSK2) are often inactivated in 2-9% of
HCC cases[2]. Overexpression of growth factors and their receptors can lead to constitutive
activation of c-Raf which consequently activate MAPK pathway[4].
Since tyrosine kinases abnormal activation have a well-established role in
hepatocarcinogenesis; they are promising targets in the management of HCC. Many multi-
tyrosine kinase inhibitors for membrane-bound and intracellular kinases were investigated in
several clinical trials for the last decade. The first-ever approved tyrosine kinase inhibitor for
advanced HCC patients is sorafenib as a first line treatment[10]. Then comes regorafenib,
multikinase inhibitor and the first drug to be approved in a decade after sorafenib as a
second line treatment for patients tolerates sorafenib but progressed over it[11][12].
Regorafenib chemical structure is based on sorafenib the bi-aryl urea compound, differs in
addition of fluorine instead of hydrogen at the phenyl group centre, hence blocked wider
range of tyrosine kinases than sorafenib[13], such as growth factor receptor kinases and the
intracellular kinases RAF an important regulator of MAPK pathway[11]. Since regorafenib
structure is based on sorafenib, it is inevitable to explore different combinatorial approaches
to protect against possible future regorafenib acquired resistance.

Berberine is a natural isoquinoline alkaloid extracted from a variety of plant families and
genera such as Coptis, and Hydrastis, but mainly as a secondary metabolite from the
genus Berberis family Berberidaceae[14]. Plants rich in berberine had been used in
traditional Chinese and Ayurvedic medicine for different purposes such as wound healing,
infectious diarrhoea and as antimicrobial, anthelmintic, and antioxidant [14], [15]. Currently,
berberine is purposed to be used to treat NASH, metabolic syndrome, management of
diabetes, obesity through lowering blood glucose, cholesterol, and LDL levels, it is marketed
as a dietary supplement after a number of clinical trials conducted to support these
uses[16]–[18].

Substantial studies have shown berberine anti tumoral activity on animal models or cell lines
of pancreatic, colorectal, breast, lung, and liver cancers[19]. Berberine exsert its antitumoral
activity via proliferation, angiogenesis, metastasis inhibition and apoptosis induction through
interacting with different molecular targets among them transcription factors, regulators of
cellular signal transductions, and cytokines[20]. Different reports attributed berberine anti-
cancer activity to modulating MAPK pathway[21], induction of mitochondrial apoptosis,
suppression of HIF-1α/VEGF signalling[22], downregulation of NF-κB[23], as well as
inhibiting AKT/mTOR pathway[24]. Target network analysis presented multiple berberine
targets among them AKT, MMP2, MMP8, interestingly molecular docking approach showed
that berberine bind to ATP-binding site of AKT thus competing with ATP and potentially
repress AKT activation[20].
Co-targeting of different molecular pathways is a suggested approach to overcome
convolution and interaction between these pathways, increasing efficiency, decreasing
toxicity, and possibly decreasing resistance. This hypothesis along with suggested inhibitory
effect of berberine on PI3K/Akt, NF-κB, and VEGF pathways in HCC pose an interesting
attempt to improve antitumor efficiency of regorafenib. So, we investigated possible long
lasting anti tumor effects of berberine and regorafenib combination in HepG2 cell line for 72
hours over regorafenib alone.
Materials and methods
Cell culture

HepG2 cells were maintained as a monolayer culture in T-25 flasks at 37°C and 5 % carbon
dioxide (CO2) grown in DMEM ( Lonza Biowhittaker™, B-4800 Verviers) supplemented with
10 % FBS (Sigma-Aldrich). Penicillin/streptomycin (Lonza Biowhittaker™) were used at a
concentration of 100 μg/ml. HepG2 cell line was obtained from the American Type Culture
Collection (ATCC, Manassas, VA, USA) via VACSERA Holding Co. for biological products
and vaccines (Agouza, Giza, Egypt). It was passaged every 2-3 days when they reached
about 80 % – 90 % confluency.

Drugs

Regorafenib (Cat no. S1178), and berberine (Cat no. S2271) were purchased from
Selleckchem (Houston, TX, USA) and.

MTT cell proliferation assay

Regorafenib and berberine were dissolved in DMSO to prepare 10mM stock solution of
each, that was then used to prepare serial dilutions of fixed ratios 1:2 in a complete growth
medium according to the calculated doses (1.25, 2.5, 5, 10, 20, 40 μM) of regorafenib and
(9.375, 18.75, 37.5, 75, 150, 300 μM) to be used in cytotoxicity analysis. Cytotoxicity for
each drug against HepG2 was determined by classic Microculture tetrazolium test (MTT). In
Brief, Cells were plated in 96 well plate at concentration of 4000 cells/well in complete media
200μl/well and incubated for 24 hr at 37 °C in 5% CO2 to allow cell attachment. Old medium
was then replaced with another 200 μL of the complete medium containing previously
prepared concentrations of each drug (three triplicates for each concentration). After addition
of the tested drugs, the plate was incubated for a further 72 hrs. Finally, MTT reagent (20 μL,
5 mg/ml) was added to the wells and incubated for 4 h. MTT treated cells were then
dissolved in 150 μL DMSO and the absorbance was recorded at 590 nm wavelength with a
reference filter of 620 nm using a microplate reader (Bio-Rad, USA). The median inhibitory
concentrations (IC50) values were calculated using the CompuSyn software (ComboSyn,
Inc).

Synergy analysis
Cells were incubated with each drug alone and in combination, using the same dose ranges
used in the former study for 72 hrs before assessment of cytotoxicity. Cell viability was
expressed as percentage relative to the vehicle control wells to calculate fold change. The
Combination Index (CI) was assessed for quantification of synergism or antagonism
between the two drugs as described by Chou-Talalay 2006 with CompuSyn software.
Moreover, Dose Reduction Index (DRI), the measure of fold-decrease of individual agent
when used in synergistic combination to achieve a given effect level compared with the
doses of each drug alone, was evaluated using the same software.

Experimental design

Three replicas of HepG2 cells received either dimethyl sulfoxide (vehicle control), Reg (12
µM), BBR (261µM), or combination of Reg (12 µM) and BBR (261µM). All experimental
procedures followed the regulatory aspects regarding the use of cell lines.

ELISA for protein analysis and quantification

Total protein was isolated from cells treated with Reg (12µM) and Ber (261µM) alone and in
combination utilizing repeated freezing/thawing cycles then centrifugation at 1000×g for 15
minutes at 2-8 °C, then was assessed using BCA method. Elisa kits used were as follows:
Human Vascular Endothelial Growth Factor (VEGF)ELISA Kit (Cusabio cat no. CSB
E11718h). Human Nuclear Factor κB P65 ELISA Kit (Mybiosource cat no. MBS040030).
Human AKT [pS473] ELISA Kit (Invitrogen cat no. KHO0111). Human p-ERK (Phospho-
Extracellular Signal Regulated Kinase) ELISA Kit (Mybiosource cat no. MBS2511875).
Human Beclin-1 (BECN-1) Elisa kit (Mybiosource cat no. MBS732891_Sandwich). Standard
curve was constructed for each protein measured to calculate its concentration in each
sample by interpolating from each curve using Curve Expert software (Hyams Development)
in U/mg protein.

Apoptosis detection
Apoptosis detection was carried out using Annexin V Alexa Fluor® 488 Apoptosis
Detection Kit II (BD Biosciences, Cat no. 556570). Cells were seeded at a density of
3.5×105 in T-25 flask and incubated for overnight at 37 °C in 5% CO2. Next day, cells were
treated with the indicated drug doses (each dose in triplicates) and after 72-hours incubation
periods, floating, and attached cells are collected and centrifuged at 2000 rpm for 5 minutes.
the cell pellets were washed twice with PBS and once with 1X binding buffer and
resuspended in 1 mL 1X binding buffer. One hundred microliter were then transferred to
clean tube and 5 μL of annexin V-FITC and 5 μL of PI were added and the tubes were
incubated at room temperature for 15 minutes in the dark. Finally, 400 μL of 1X binding
buffer were added and the cells were analysed using flow cytometer (BD FACSCalibur, BD
Biosciences).

Western blotting analysis

To evaluate combination effects after cell treatment, cell cycle and autophagy pathways
were analyzed by Western blotting analysis. After harvest, the cells were washed twice with
cold phosphate-buffered saline (PBS) and then lysed in RIPA buffer (Sigma-Aldrich, Milan;
Italy). The total soluble proteins were subjected to Western blotting analysis. Protein
concentration was determined using Micro BCA Protein Assay kit (Thermo Scientific,
Rockford, IL USA) as described in manufacture manual. Equal amounts of protein (20 μg)
were resolved on SDS–PAGE and transferred to polyvinyldifluoride (PVDF) filters (BioRad,
Milan; Italy). The filters were blocked 5% (w/v) BSA for 2 h at room temperature and then
probed with primary antibody overnight at 4°C. The primary antibodies were directed against
the following proteins: Beclin-1 (52 kDa), and β-actin (42 kDa) (Santa Cruz Biotechnology,
Santa Cruz, CA; USA). After three washes, incubation was followed by reaction with
horseradish peroxidase-conjugated specific secondary antibody for 1 h at room temperature
and the bands were visualized by enhanced chemiluminescence detection system (Bio-
Rad). The primary antibodies against proteins were removed by stripping buffer (Thermo
Fisher Scientific, MA, USA) and membrane was re-probed with the antibody against β-actin.

Densitometric values were normalized using β-actin and analysed with ImageJ freeware
by Wayne Rasband from National Institute of Health (USA).

Statistical analysis

All results obtained were presented as mean ± SEM. GraphPad Prism version 9 for
Windows (GraphPad Software) was used for statistical analysis. To compare between
experimental groups, one-way analysis of variance test (one-way ANOVA), followed by
Bonferroni’s multiple comparison test. Differences with values of P<0.05 were considered
statistically significant.
Results
IC50 of Reg and BBR in HepG2 cell line

In order to measure growth inhibition effect of Regorafenib, berberine alone and analyse
effect of their interaction, serial concentrations with a fixed 1:2 ratio of drugs were used to
treat HepG2 cells for 72 hours. Half maximal inhibitory concentration (IC50) of each drug
was calculated using dose response curve to be implicated in successive experiments.

The effect of Reg and/or BBR on the growth of HepG2 cells is presented in (Figure 1).
Treatment with regorafenib in a concentration range of (1.25-40 μM) caused dose-
dependent growth inhibition with IC50 of 12.027 μM (Figure 1a). In the same context,
berberine treatment was applied within range (9.375-300 μM) resulted in a dose-dependent
cytotoxicity with IC50 of 261.104 μM (Figure 1b). While treatment of HepG2 cells with
combination of the two drugs with the same concentration gradients of each alone is shown
in (Figure 1c).

Combination index (CI) and dose reduction index (DRI) of REG and BBR

To evaluate the significance of using berberine in combination with regorafenib on HepG2

cells, combination index (CI) and dose reduction indices (DRIs) were determined using
CompuSyn software employing data acquired from MTT assay, to identify the type of
interaction between the two drugs. CI value of Reg and Ber combination at 50% fractional
growth inhibition (Fa=0.5) was 0.918, concluding synergistic activity between the two drugs
as CI<1. Treatment of HepG2 with combination lead to suppression of cell viability with IC50
of 69.76 μM (61.557 μM for berberine + 8.207 μM for regorafenib). To determine possibility
of dose reduction of each drug in the combination group, DRI was assessed and found to be
more than 1, results are elucidated in (Figure 2 or Table 1) where Reg dose required to
induce 50% fractional growth inhibition alone was 12.027 µM whereas in combination
concentration dropped to 8.207 µM meaning that dose was reduced 1.46-fold. Berberine
likewise has a DRI of 4.2 as its concentration at Fa=0.5 when used alone was 261.104 µM,
where in combination its required dose dropped to 61.557 µM.

REG and/or BBR treatment reduced the phosphorylated Akt (S473)

p-Akt protein level was measured quantitively in HepG2 cell lysate (U/mg protein). Treatment
with regorafenib and berberine each alone significantly decreased p-Akt protein level
compared to control group by 32.5% and 31.81% respectively as shown in (Figure 3).
Treating HepG2 cells with the combination of the two drugs caused more substantial
reduction in p-Akt protein levels by 45.29% compared to control with significant inhibition
(P<0.05) compared to each drug alone.

REG and/or BBR treatment reduced the phosphorylated ERK (S473)

p-ERK protein levels (Pg/mg protein) was measured quantitevly in HepG2 cell lysate to
determine the degree of inhibition of this pathway. Results demonstrated in (Figure 4)
showing that regorafenib and berberine treatment each alone significantly decressed p-ERK
protein levels compared to control group by 40.17% and 29.9% respectively. Treating
HepG2 cells with the combination triggered further signifcant reduction in p-ERK protein
levels by 47.6% compared to control with significant reduction compared each drug alone.

REG and/or BBR treatment inhibited NF-κB p65

NF-κB protein level was measured quantitively (ng/mg protein) in cell lysate of HepG2 cells.
Results depicted in (Figure 5) indicates that treatment of cells with regorafenib reduced NF-
κB protein level significantly by 59.83% and berberine by 65.32% each alone compared to
control cells. While with combination the degree of NF-κB protein level reduction was also
significant compared to each drug alone, and even more significant reduction than control by
77.91%.

REG and/or BBR treatment reduced VEGF protein level

VEGF as a major angiogenesis modulator was measured quantitatively in cell lysate of

control and treated HepG2 cells to reveal the following results illustrated in (Figure 6). These
results show that regorafenib, berberine each alone induced significant reduction of VEGF
levels (Pg/mg cellular protein) in comparison to untreated control group. VEGF protein
expression was reduced by 52% in Reg-treated group, and 58.6% in Ber-treated group.

Remarkably, cells treated with the combination showed more decline of VEGF protein level
compared to untreated cells by 80.6%. Its inhibition was also significant compared to cells
treated with each drug alone.

Effect of REG and/or BBR on autophagy marker Beclin-1 protein expression

Beclin-1 protein level in HepG2 cell lysate was measured by relative quantification using
western blotting, result is illustrated in (Figure 7) Highest concentration of beclin-1 was in
control group, while regorafenib treatment reduced beclin-1 protein level significantly by
29.4%, and berberine by 44.76% compared to control group.
 Additively, combination treated groups showed more reduction in Beclin-1 protein level
compared to control groups by 76.39% with significant reduction compared to each drug
alone. Fold change of protein expression between each treated group and control is
illustrated in (Table 1)

REG and/or BBR treatment induced apoptosis in HepG2 cell line

Results indicate that in control group (Figure 8a) 3.90% of cells were in early apoptotic stage
while 2.50% of cells were in late apoptotic stage, 6.85% of cells were already dead and lastly
85.84% of control cells were alive. While cells treated with regorafenib (Figure 8b) had
7.24% of cells in early apoptotic stage, 4.51% of cells were in late apoptotic stage, 19.75%
of cells were dead and lastly 68.55% of the cells were still alive.

Whereas berberine treated group (Figure 8c) showed more apoptotic cells in early stage
12.03%. and 11.54% of cells were in late apoptotic stage, 25.51% of cells were necrotic, and
50.91% of cells were alive. Regarding combination treated group (Figure 8d) 7.41% of cells
were early apoptotic, much more cells were in late apoptotic stage19.58% of cells, 39.09%
of population were necrotic, and 32.49% of combination treated group were alive.

Chart in Figure 9 shows effect of treatments on apoptosis of HepG2 cells in different stages,
where Combined treatment of berberine plus regorafenib caused significant increase in
population of late apoptotic and dead cells compared to treated group and to each drug
alone, and significant reduction in living cell population. In the same context, berberine
treated group showed significant induction in population of early, late apoptotic, and dead
cells compared to control group, as to regorafenib, it showed significant increase in
population of necrotic cells compared to control. Both of regorafenib and berberine treated
groups showed significant repression of viable cells compared to control.

Effect of REG and/or BBR on CyclinD1 protein Level

To determine tested drugs’ ability to hinder tumor proliferation and induce cell cycle arrest,
Cyclin D1 as a required protein for the cell to progress through G1 phase of the cell cycle to
the DNA synthesis phase (S phase)[25], was measured in HepG2 cell lysate of control and
treated groups. Results shown in (Figure 10) illustrates that regorafenib as well as berberine
significantly reduced Cyclin D1 (ng/mg protein) protein levels by 52.97% and 63.86%
respectively, compared to control groups.

As for cells treated with combined treatment eminently reduced expression of Cyclin D1 by
81.68% in comparison with control with superior inhibition effect compared to berberine
treated, and regorafenib treated cells.
Discussion
Accumulative evidences showed limitation of clinical efficacy of single agent or inhibitor for
one pathway due to increased crosstalk between pathways, and feedback regulation that
may lead to drug resistance [26]. Crosstalk between PI3K/Akt pathway, JAK/STAT and
MAPK pathway during sorafenib treatment was associated with sorafenib acquired
resistance in HCC cell lines [27], which was restored by targeting Akt [28]. In the same
context, Ras/MAPK pathway deregulation is considered a determinant in cellular resistance
to PI3K inhibitors[29].

Results of our study showed enhanced antitumor activity of regorafenib by combination with
berberine by co targeting multiple pathways including PI3K/Akt, MAPK and NF-κB pathways
in human HepG2 cell line. Synergistic effect of this combination was seen at cellular level as
confirmed by cell viability assay, combination index and dose reduction index, and on
molecular level indicated by significant inhibition of CD1, VEGF, Beclin1, and induction of
apoptosis. Combination also showed downregulation of: MAPK pathway indicated by
inhibition of p-ERK, PI3K/Akt/mTOR/NF-κB pathway indicated by inhibition of p-Akt and NF-
κB p65.

Regorafenib is a multityrosine kinase inhibitor that exert its antitumor activity by targeting
kinases that regulate different signalling pathways in tumor cells and tumor
microenvironment such as growth factor receptor kinases Tie-2, VEGFR1,2,3, PDGFR,
FGFR1,2 involved in tumor and endothelial cell angiogenesis, proliferation, and migration.
Regorafenib can also block oncogenic membrane kinases such as KIT, and RET involved in
tumor growth promotion and intracellular kinases such as C-RAF/RAF-1, wild type BRAF
and mutant BRAFV600E key signalling molecule in the RAS/RAF/MAPK pathway[11]. As a
Raf inhibitor, it can cause inhibition of downstream kinases such as ERK. In current study,
regorafenib showed significant p-ERK inhibition compared to control which is in consistence
with earlier observations in SK-Hep1 cells[30], MDA-MB-231, BxPC-3 and LOX cell lines[11],
and in HCT 116 and HT-29 colon cancer cell lines[31].

Inhibition of upstream kinases such as growth factor receptors involved in activation of

PI3K/Akt/mTOR cascade can contribute to indirect marginal inhibition of p-Akt, as shown per
our results, this finding is consistent with earlier studies where regorafenib inhibited Akt
phosphorylation on HepG2 and Hep3B cells[32] and on HT29 cell line[33].
Berberine has multiple protein targets in the cell including signalling cassettes, transcription
and translation involved proteins due to its intercalating- like structure [34]. Different attempts
were made to determine some of its molecular targets using molecular docking and protein-
protein interaction network. L. Song and colleagues were able to determine Akt as one of its
direct targets and using molecular docking they were able to demonstrate that berberine
bind to Akt active site and further inhibit its phosphorylation and activation on HepG2 and
MHCC97-H[20]. PJ Kaboli and colleagues performed molecular docking and molecular
dynamics simulations for members of the MAPK pathway starting from EGFR, P38, and
ERK. Their experiments predicted that berberine have inhibitory effect on EGFR and ERK
with lower inhibition of P38 activity[34].

This berberine ability to significantly inhibit p-Akt, and p-ERK was observed in our results, in
context with previous reports showed that berberine inhibited p-Akt protein expression in EU-
6 acute lymphocytic leukemia cell line[35], and SW480 cell line [36]. In our study results,
berberine compared with regorafenib caused moderate yet significant inhibition of ERK
phosphorylation In accordance with former research where berberine attenuated p-ERK
levels in MGC 803 gastric cancer cell line in a time and dose dependant manner and
confirmed that proliferation inhibition is established through deactivating ERK hence MAPK
pathway[37], Berberine also suppressed p-ERK in Cholangiocarcinoma cell line[38].
Although other research reported that in a much lower dose of berberine for 24h in A549
lung adenocarcinoma cell line it caused induction on ERK phosphorylation[39]. So, berberine
effect on MAPK/ERK pathway is dependent on cell type.

Present study showed that co treatment of berberine with regorafenib caused significant
reduction of p-Akt and p-ERK levels than regorafenib alone, confirming the synergistic effect
of combination in dual targeting of the two pathways.

NF-κB plays an important role in liver cancer progression, it is overexpressed in up to 70% of

HCC cases with significantly higher expression compared to adjacent normal liver tissue[40]
[41]. In vitro studies showed that NF- κB signalling pathway participates in linking different
regulatory pathways of HCC, where its activation can stimulate proliferation, angiogenesis,
invasion and antitumor resistance[42]. It triggers chemokine release that attracts different
immune cells such as natural killer cells, monocytes, T and B cells, that exacerbate
inflammatory environment[40]. Thus its activation triggers inflammatory process , enhanced
proliferation, angiogenesis, metastasis, invasion, and drug resistance[40].
In normal physiologic condition NF-κB dimers bound to IκB proteins (Inhibitors of NF-κB) and
remained in the cytoplasm in inactive form. For NF-κB activation, IκB are phosphorylated by
IKK (IκB kinase) that leads to their proteasomal degradation thus release of NF-κB and its
nuclear translocation to exert its activity[43]. Akt can regulate NF-κB through stimulation of
IKK by phosphorylation of IKKα subunit directly[44] or by activation of Tpl-2 (tumor
progression locus 2) that in turn phosphorylate IKKβ subunit[45] consequently activate IKK
mediated NF-κB stimulation.

Current study showed significant reduction in NF-κB p65 activation along with p-Akt
inhibition which suggest downregulation of PI3K/AKT/NF-κB pathway. Observed inhibition of
p-Akt by regorafenib and berberine could infer an explanation to NF-κB inhibition through its
activation repression by IKK. Akt- modulated NF-κB inhibition after regorafenib treatment is
observed in CL1-5-F4 lung cancer cell line[46], H. Goto and colleagues also confirmed that
berberine inhibit NF-κB activation through upstream inhibition of IKK, IκB phosphorylation
and inactive IκB accumulation in PEL( Primary Effusion Lymphoma) cell lines [47]. NF- κB
phosphorylation inhibition after berberine treatment is consistent with earlier experiments
performed on HepG2 cell line[48],[23] and B16F‐10 melanoma cells[49]

NF-κB p65 inhibition could also be attributed to p-ERK inhibition as earlier research
confirmed hypoxia induced acidosis increase NF- κB activation through ERK
phosphorylation in 22Rv1 prostate cancer cell line[50], this correlation between p-ERK and
NF-κB has also been shown in SK-HEP-1 cells when treated with regorafenib or p-ERK
inhibitor showed significant deactivation of NF-κB suggesting that regorafenib inhibition
occurred through ERK dephosphorylation[30].

Activation of all beforementioned pathways contribute to diminished apoptotic cell death and
increased proliferation, downregulation of these pathways using suggested combination led
to induction of apoptotic cell death. Induction of apoptotic cell death by berberine could be
attributed to proven downregulation of PI3K/Akt pathway that leads to induction of BAX and
PARP expression[51].It also could be referred to inhibition of ERK activation that may confer
a role in apoptotic induction[52]. These finding are in the same context with earlier finding of
berberine causing induction of cell death in Huh7 liver cancer cell line[53] and in MGC803
gastric cancer cell line[54]
 Using regorafenib in combination with berberine enhanced its anti-apoptotic activity, as
combination treated group increased the dead/ apoptotic portions of population more than
regorafenib alone overcoming regorafenib induced quiescence in which cells became
dormant neither growing nor detaching as observed in earlier reports [55], [56] , this
observation is noteworthy because in clinical application patients undergo multikinase
inhibitors require therapy breaks, in which tumor cells treated with regorafenib may became
dormant, and that is why using combination is more rationale. Increased population of
apoptotic and dead cells in combination treated group in comparison with each drug alone or
with control, could infer crosstalk between different signalling pathways inhibited by the two
drugs. PI3K/Akt/mTOR pathway inhibition can impact apoptosis through downregulation of
MDM2 in turn activate p53 and inhibit Bcl2[57]. NF-κB pathway inhibition can also affect
apoptosis by modulating C-FLIP and MCL-1[23], [30].

Angiogenesis, a well-known hallmark of cancer that solid tumor rely on for survival, growth,
sprouting and metastasis using different angiogenic and growth factors redundant in tumor
microenvironment [58]. Hypoxia is considered as stimulator of autocrine VEGF expression,
through Hypoxia inducible factor (HIF-1α) transcriptional regulation of many growth factors
including VEGF[59]. HIF-1α protein synthesis can be induced through phosphorylation of
eukaryotic translation initiation factor 4E (eIF-4E) by either mTOR after activation of
PI3K/Akt/mTOR pathway, or ERK after activation of RAS/RAF/ERK pathway[60].

So, reduction of VEGF protein expression in Reg-treated group can be attributed to its well
established role in inhibition of receptor tyrosine kinases that subsequently inhibits its
downstream signalling cascade in addition to inhibition of RAF kinase which also contribute
to regulation of VEGF expression[61], and dephosphorylation of eIF-4E as observed in
endometrial adenocarcinoma cell lines[62]

Berberine effect on VEGF reduction is also consistent with previous researches

demonstrated that it suppressed HIF1α protein synthesis in KM12C colon cancer cell line by
inhibiting mTOR phosphorylation [63] and repressed VEGF at transcriptional level in HepG2
cell line[22], suggesting that berberine inhibit VEGF production via inhibiting mTOR-
dependent HIF1α protein expression. Increased reduction in VEGF expression in
combination treated group reflects effect of the two drugs combined on inhibiting multiple
pathways through tumor cells.
Unsurprisingly, Cyclin D1 is overexpressed frequently in different primary tumors, activation
mutations in CCND1 presents in 4-6 % of HCC[2]. This overexpression is a result of DNA
damage and accumulation of chromosomal abnormalities, that leads to CD1 build up in
nuclei and interfering with cell cycle regulation[64].

PI3K/Akt pathway plays pivotal role in cell cycle regulation. Upon Akt activation it regulates
various cell cycle progression substrates related to G1/S transition. This regulation could be
indirect via inhibition of GSK3β that phosphorylate Cyclin D1 at Thr286 to induce its
proteolysis[65][66]. Once p-Akt is inhibited by berberine, regorafenib or combination as
found per our results, GSK3β is activated and CD1 proteolysis is triggered leading to cell
cycle arrest.

NF-κB can regulate Cyclin D1 on transcriptional level as it serves as a transcription factor of

many oncogenes including Cyclin D1. Upon translocation to nucleus, it binds to its specific
binding site of CD1 promoter to aid initiating its transcription that eventually led to cell cycle
progression[67]. Our tested drugs proved NF-κB inhibition abilities could represent another
explanation of Cyclin D1 inhibition.

These results are accordant with previous findings where berberine suppressed CD1
through post translational proteasomal degradation that was observed in HepG2 cell
lines[68], and through inhibition of NF-κB observed in KKU-213 cholangiocarcinoma cell
line[38]. Regorafenib effect on CD1 inhibition was previously described in different HCC cell
lines (HepG2-HUH7- HEP3B) [69],[70], and in U-2 OS and MG-63 osteosarcoma cell
lines[71].

Autophagy possesses a controversial role in cancer biology and therapeutics. It is first

described as essential player in cell survival under starvation and stressful environment, yet
its role in cancer is still debateable, it is considered a double-edged sword where its activity
is relying on the stage of tumor progression, cellular genetic background, and what factor
stimulated it, so that its activation may lead to different outcome depending on context[72]
As cancer increasingly seen as metabolic disease, autophagy as a metabolic stress
modulator is currently recognized as vital target for anti-cancer therapy, indeed many
anticancer treatments affects autophagy either by induction or attenuation that may led to
anti-tumor drug resistance or intensify anti-tumor effect. Highly developed tumours often
suffer from nutrients deprivation , metabolic stress and hypoxia, anti tumor treatments act as
another stressful condition imposed upon tumor cell, that is when autophagy role come to
degrade macromolecules to ease metabolic stress and maintain cell survival[73]. This
approach suggests that autophagy inhibition could pose advantageous tumor survival effect.

To explore effect of regorafenib and berberine on autophagy, Beclin-1 protein expression

level was measured to reach a better understanding of how this process influence the
established anti tumor effect of our drugs. Beclin-1 is one of the core modulators of
autophagy, as it is an element in the Beclin1-PI3KC complex that is essential in
autophagosome formation, it is tightly regulated by many cellular factors including its binding
partners in the complex[74].

Regorafenib and berberine each alone significantly abrogated beclin-1 expression, this
abrogation was even improved in combination treatment. With respect to our drugs
forementioned impact on increasing cell death significantly, this reduction of autophagy
marker should be in consistence with autophagy as tumor progression activator approach. In
the same time, our treatments also inhibited Akt/mTOR pathway which is a known inhibitor
of autophagy, yet research stated that it is not the only pathway involved on its regulation,
instead it is regulated jointly by multiple pathways as it can be regulated by different inputs
such as growth factors, cytokines or level of nutrients in the cell [75].

NF-κB is another regulator of autophagy, it has been shown to induce Beclin-1 gene
expression by binding to Beclin-1’s promoter in T cells[72], and Mantle cell lymphoma cells
[76]. IKK complex is also capable of inducing autophagy genes[77]. Noteworthy, activation of
beclin-1 mediated autophagy led to constitutive activation of NF-κB and STAT-3 mediated
induction of different cytokines and IL-6 among other factors involved in survival and
metastasis of HUVEC starved cells and HTLV-1 cells[78], [79]. Same results of NF-κB
attenuation occurred after silencing beclin-1 gene to further confirm this finding[78]. These
findings also support our approach and in consistence with our findings of tested drugs effect
on NF-κB protein level decline, with combination causing the highest beclin-1 and NF-κB
reduction.
Although earlier research suggested that berberine induce autophagy marker beclin-1 with
simultaneous apoptosis induction using HepG2 cells or EU-6 acute lymphoblastic leukemia
cell lines, yet upon closer inspection of these research we found that berberine caused
beclin-1 induction on HepG2 cells either after 6h treatment using 50-200μM of berberine
while apoptosis tests were performed after12h incubation[80] or after 24 h[81]. As for EU-6,
beclin-1 induction occurred using 100μM of berberine for 24h[35]. Whereas apoptosis
induction and autophagy inhibition in our study was performed in the same timeline after 72h
incubation with 261μM.

This suggests that role of autophagy as tumor suppressor or promoter indeed is time and
dose dependant manner, as in first encounter of tumor cell with cytotoxic drugs, it activate
autophagy cytoprotective mechanism in attempt to survive and delays rate of drug-induced
apoptosis, while longer incubation periods or higher doses eventually led to autophagy
inhibition. This could by resultant from commitment of cells to apoptosis where autophagy
protection is not desirable anymore[82][83].

Different research suggested that autophagy reduction led to re-sensitization of previously

resistant tumor cells after combining with autophagy inhibitors. For instance, in HepG2 cells
previously treated with etoposide and showed increased autophagy markers, the use of
beclin-1 siRNA boosted etoposide- induced apoptosis[84]. This effect was also observed in
colon cancer xenografts using chloroquine to re-sensitize tumor cells to Vorinostat [85].

Autophagy induced resistance was also observed in regorafenib close relative, sorafenib.
Many studies found that autophagy is induced by sorafenib, most of them indicated that this
induction is a survival mechanism as response to stress not anti tumor mechanism of action.
This hypothesis was confirmed through either knockdown of autophagy markers such as
ATG7 siRNA on Huh7 [86], beclin-1 knockdown in FTC133 thyroid cancer cells [87], or using
pharmacologic autophagy inhibitors such as chloroquine in FTC133 cells [87] or 3-
Methyladenine in different human hepatoma cells [87] all of them led to induction of
apoptosis and reversed sorafenib resistance. Our results indicate a successful combination
capable of HCC eradication and protection from any forthcoming autophagy-induced
regorafenib resistance.

In conclusion, combining regorafenib and berberine lead to mutual dose reduction with
synergistic inhibitory effect on HepG2 cells viability, which could be attributed to inhibition of
signalling pathways such as PI3K/Akt/mTOR/ NF-κB and MAPK pathways involved in
various oncogenic processes. This was reflected on carcinogenesis hallmarks demonstrated
herein such as enhanced apoptosis, and cell cycle regression. Angiogenesis inhibition and
autophagy reduction, where autophagy is supposed to be tumor cytoprotective to help
cancer cells survive under stress conditions. Although, autophagy activity varies between
different tissues and different stages of carcinogenesis, but a threshold maintains balance
between moderate autophagy that may cause cell death resistance and excessive
autophagy that led to cell death is context specific such as many cellular modulators in
cancer. This indicates that this combination can be considered a promising candidate for
additional preclinical and clinical exploration to further enhance HCC management.
[1] H. Sung et al., “Global Cancer Statistics 2020: GLOBOCAN Estimates of
Incidence and Mortality Worldwide for 36 Cancers in 185 Countries,” CA.
Cancer J. Clin., vol. 71, no. 3, pp. 209–249, May 2021, doi:
https://doi.org/10.3322/caac.21660.

[2] M. Allaire and J.-C. Nault, “Molecular targets for HCC and future treatments,”
J. Hepatol., vol. 66, no. 1, pp. 234–235, Jan. 2017, doi:
10.1016/j.jhep.2016.07.034.

[3] A. Alqahtani, Z. Khan, A. Alloghbi, T. S. Said Ahmed, M. Ashraf, and D. M.

Hammouda, “Hepatocellular Carcinoma: Molecular Mechanisms and Targeted
Therapies,” Medicina , vol. 55, no. 9. 2019, doi: 10.3390/medicina55090526.

[4] M. Dimri and A. Satyanarayana, “Molecular Signaling Pathways and

Therapeutic Targets in Hepatocellular Carcinoma,” Cancers , vol. 12, no. 2.
2020, doi: 10.3390/cancers12020491.

[5] K. Breuhahn, T. Longerich, and P. Schirmacher, “Dysregulation of growth

factor signaling in human hepatocellular carcinoma,” Oncogene, vol. 25, no.
27, pp. 3787–3800, 2006, doi: 10.1038/sj.onc.1209556.

[6] J. Halaschek-Wiener, V. Wacheck, Y. Kloog, and B. Jansen, “Ras inhibition

leads to transcriptional activation of p53 and down-regulation of Mdm2: two
mechanisms that cooperatively increase p53 function in colon cancer cells.,”
Cell. Signal., vol. 16, no. 11, pp. 1319–1327, Nov. 2004, doi:
10.1016/j.cellsig.2004.04.003.

[7] B. Akhtar, F. Muhammad, A. Sharif, M. F. Akhtar, and W. Majeed, “Diverse

Signaling Pathways and Current Status of Molecular Targeted Treatments for
Hepatocellular Carcinoma.,” Crit. Rev. Eukaryot. Gene Expr., vol. 27, no. 4, pp.
373–385, 2017, doi: 10.1615/CritRevEukaryotGeneExpr.2017021006.

[8] L. Zhou, Y. Huang, J. Li, and Z. Wang, “The mTOR pathway is associated with
the poor prognosis of human hepatocellular carcinoma.,” Med. Oncol., vol. 27,
no. 2, pp. 255–261, Jun. 2010, doi: 10.1007/s12032-009-9201-4.

[9] R. Allen and D. Li, “Hepatocellular Carcinoma BT - Oncology in the Precision

Medicine Era: Value-Based Medicine,” R. Salgia, Ed. Cham: Springer
International Publishing, 2020, pp. 259–271.

[10] L. Lang, “FDA Approves Sorafenib for Patients With Inoperable Liver Cancer,”
Gastroenterology, vol. 134, no. 2, p. 379, Feb. 2008, doi:
10.1053/j.gastro.2007.12.037.

[11] S. M. Wilhelm et al., “Regorafenib (BAY 73-4506): a new oral multikinase

inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with
potent preclinical antitumor activity.,” Int. J. cancer, vol. 129, no. 1, pp. 245–
255, Jul. 2011, doi: 10.1002/ijc.25864.

[12] J. Bruix et al., “Regorafenib for patients with hepatocellular carcinoma who
progressed on sorafenib treatment (RESORCE): a randomised, double-blind,
 placebo-controlled, phase 3 trial.,” Lancet (London, England), vol. 389, no.
10064, pp. 56–66, Jan. 2017, doi: 10.1016/S0140-6736(16)32453-9.

[13] G. Goel, “Evolution of regorafenib from bench to bedside in colorectal cancer:

Is it an attractive option or merely a ‘me too’ drug?,” Cancer Manag. Res., vol.
10, pp. 425–437, 2018, doi: 10.2147/CMAR.S88825.

[14] M. A. Neag et al., “Berberine: Botanical Occurrence, Traditional Uses,

Extraction Methods, and Relevance in Cardiovascular, Metabolic, Hepatic, and
Renal Disorders,” Front. Pharmacol., vol. 9, p. 557, Aug. 2018, doi:
10.3389/fphar.2018.00557.

[15] J. Yin, H. Zhang, and J. Ye, “Traditional chinese medicine in treatment of

metabolic syndrome,” Endocr. Metab. Immune Disord. Drug Targets, vol. 8,
no. 2, pp. 99–111, Jun. 2008, doi: 10.2174/187153008784534330.

[16] J. Yin, H. Xing, and J. Ye, “Efficacy of berberine in patients with type 2
diabetes mellitus,” Metabolism., vol. 57, no. 5, pp. 712–717, May 2008, doi:
10.1016/j.metabol.2008.01.013.

[17] H. Wang, C. Zhu, Y. Ying, L. Luo, D. Huang, and Z. Luo, “Metformin and
berberine, two versatile drugs in treatment of common metabolic diseases,”
Oncotarget, vol. 9, no. 11, pp. 10135–10146, Sep. 2017, doi:
10.18632/oncotarget.20807.

[18] H.-M. Yan et al., “Efficacy of Berberine in Patients with Non-Alcoholic Fatty
Liver Disease,” PLoS One, vol. 10, no. 8, pp. e0134172–e0134172, Aug. 2015,
doi: 10.1371/journal.pone.0134172.

[19] S. Hu, R. Zhao, Y. Liu, J. Chen, Z. Zheng, and S. Wang, “Preventive and
Therapeutic Roles of Berberine in Gastrointestinal Cancers,” Biomed Res. Int.,
vol. 2019, p. 6831520, 2019, doi: 10.1155/2019/6831520.

[20] L. Song et al., “Exploring the active mechanism of berberine against HCC by
systematic pharmacology and experimental validation,” Mol Med Rep, vol. 20,
no. 5, pp. 4654–4664, 2019, doi: 10.3892/mmr.2019.10698.

[21] M. Jie et al., “Systematic Investigation of Berberine for Treating Hepatocellular

Carcinoma Based on Network Pharmacology,” Digit. Chinese Med., vol. 2, no.
3, pp. 127–135, 2019, doi: https://doi.org/10.1016/j.dcmed.2019.12.001.

[22] S. Jie et al., “Berberine inhibits angiogenic potential of Hep G2 cell line through
VEGF down-regulation in vitro.,” J. Gastroenterol. Hepatol., vol. 26, no. 1, pp.
179–185, Jan. 2011, doi: 10.1111/j.1440-1746.2010.06389.x.

[23] M. Li et al., “Induction of Apoptosis by Berberine in Hepatocellular Carcinoma

HepG2 Cells via Downregulation of NF-κB.,” Oncol. Res., vol. 25, no. 2, pp.
233–239, Jan. 2017, doi: 10.3727/096504016X14742891049073.

[24] Y. Kou et al., “Berberine suppressed epithelial mesenchymal transition through

cross-talk regulation of PI3K/AKT and RARα/RARβ in melanoma cells,”
Biochem. Biophys. Res. Commun., vol. 479, no. 2, pp. 290–296, 2016, doi:
 https://doi.org/10.1016/j.bbrc.2016.09.061.

[25] R. Donnellan and R. Chetty, “Cyclin D1 and human neoplasia.,” Mol. Pathol.,
vol. 51, no. 1, pp. 1 LP – 7, Feb. 1998, doi: 10.1136/mp.51.1.1.

[26] Q. Zhou, V. W. Y. Lui, and W. Yeo, “Targeting the PI3K/Akt/mTOR pathway in

hepatocellular carcinoma,” Futur. Oncol., vol. 7, no. 10, pp. 1149–1167, Oct.
2011, doi: 10.2217/fon.11.95.

[27] Y.-J. Zhu, B. Zheng, H.-Y. Wang, and L. Chen, “New knowledge of the
mechanisms of sorafenib resistance in liver cancer.,” Acta Pharmacol. Sin.,
vol. 38, no. 5, pp. 614–622, May 2017, doi: 10.1038/aps.2017.5.

[28] K.-F. Chen et al., “Activation of phosphatidylinositol 3-kinase/Akt signaling

pathway mediates acquired resistance to sorafenib in hepatocellular
carcinoma cells.,” J. Pharmacol. Exp. Ther., vol. 337, no. 1, pp. 155–161, Apr.
2011, doi: 10.1124/jpet.110.175786.

[29] K. Yu, L. Toral-Barza, C. Shi, W.-G. Zhang, and A. Zask, “Response and
determinants of cancer cell susceptibility to PI3K inhibitors: combined
targeting of PI3K and Mek1 as an effective anticancer strategy.,” Cancer Biol.
Ther., vol. 7, no. 2, pp. 307–315, Feb. 2008, doi: 10.4161/cbt.7.2.5334.

[30] J.-J. Tsai, P.-J. Pan, and F.-T. Hsu, “Regorafenib induces extrinsic and
intrinsic apoptosis through inhibition of ERK/NF-κB activation in hepatocellular
carcinoma cells,” Oncol Rep, vol. 37, no. 2, pp. 1036–1044, 2017, doi:
10.3892/or.2016.5328.

[31] K. Matsuoka, F. Nakagawa, N. Tanaka, H. Okabe, K. Matsuo, and T. Takechi,

“Effective Sequential Combined Chemotherapy with Trifluridine/Tipiracil and
Regorafenib in Human Colorectal Cancer Cells,” Int. J. Mol. Sci., vol. 19, no.
10, p. 2915, Sep. 2018, doi: 10.3390/ijms19102915.

[32] R. Han and S. Li, “Regorafenib delays the proliferation of hepatocellular

carcinoma by inducing autophagy.,” Pharmazie, vol. 73, no. 4, pp. 218–222,
Apr. 2018, doi: 10.1691/ph.2018.7988.

[33] Y.-C. Liu, J.-J. Tsai, Y.-S. Weng, and F.-T. Hsu, “Regorafenib suppresses
epidermal growth factor receptor signaling-modulated progression of colorectal
cancer,” Biomed. Pharmacother., vol. 128, p. 110319, 2020, doi:
https://doi.org/10.1016/j.biopha.2020.110319.

[34] P. Jabbarzadeh Kaboli, M. P.-Y. Leong, P. Ismail, and K.-H. Ling, “Antitumor
effects of berberine against EGFR, ERK1/2, P38 and AKT in MDA-MB231 and
MCF-7 breast cancer cells using molecular modelling and in vitro study,”
Pharmacol. Reports, vol. 71, no. 1, pp. 13–23, 2019, doi:
10.1016/j.pharep.2018.07.005.

[35] J. Liu et al., “Berberine Induces Autophagic Cell Death in Acute Lymphoblastic
Leukemia by Inactivating AKT/mTORC1 Signaling,” Drug Des. Devel. Ther.,
vol. 14, pp. 1813–1823, May 2020, doi: 10.2147/DDDT.S239247.
[36] G. Li, C. Zhang, W. Liang, Y. Zhang, Y. Shen, and X. Tian, “Berberine
regulates the Notch1/PTEN/PI3K/AKT/mTOR pathway and acts synergistically
with 17-AAG and SAHA in SW480 colon cancer cells,” Pharm. Biol., vol. 59,
no. 1, pp. 21–30, Dec. 2021, doi: 10.1080/13880209.2020.1865407.

[37] H. Li, H. Wu, B. Zhang, H. Shi, and X. Wu, “MAPK pathways are involved in
the inhibitory effect of berberine hydrochloride on gastric cancer MGC 803 cell
proliferation and IL-8 secretion in vitro and in vivo,” Mol Med Rep, vol. 14, no.
2, pp. 1430–1438, 2016, doi: 10.3892/mmr.2016.5361.

[38] N. Puthdee et al., “Berberine Induces Cell Cycle Arrest in Cholangiocarcinoma

Cell Lines via Inhibition of NF-κB and STAT3 Pathways,” Biol. Pharm. Bull.,
vol. 40, no. 6, pp. 751–757, 2017, doi: 10.1248/bpb.b16-00428.

[39] F. Zheng et al., “p38α MAPK-mediated induction and interaction of FOXO3a

and p53 contribute to the inhibited-growth and induced-apoptosis of human
lung adenocarcinoma cells by berberine,” J. Exp. Clin. Cancer Res., vol. 33,
no. 1, p. 36, 2014, doi: 10.1186/1756-9966-33-36.

[40] M.-Y. Wu, G.-T. Yiang, P.-W. Cheng, P.-Y. Chu, and C.-J. Li, “Molecular
Targets in Hepatocarcinogenesis and Implications for Therapy,” J. Clin. Med.,
vol. 7, no. 8, p. 213, Aug. 2018, doi: 10.3390/jcm7080213.

[41] E. Pikarsky et al., “NF-kappaB functions as a tumour promoter in inflammation-

associated cancer.,” Nature, vol. 431, no. 7007, pp. 461–466, Sep. 2004, doi:
10.1038/nature02924.

[42] G. He and M. Karin, “NF-κB and STAT3 – key players in liver inflammation and
cancer,” Cell Res., vol. 21, no. 1, pp. 159–168, 2011, doi:
10.1038/cr.2010.183.

[43] M. I. KÖRBER, A. STARIBACHER, I. N. A. RATZENBÖCK, G. STEGER, and

R. M. MADER, “NFκB-Associated Pathways in Progression of
Chemoresistance to 5-Fluorouracil in an In Vitro Model of Colonic Carcinoma,”
Anticancer Res., vol. 36, no. 4, pp. 1631 LP – 1639, Apr. 2016, [Online].
Available: http://ar.iiarjournals.org/content/36/4/1631.abstract.

[44] D. Bai, L. Ueno, and P. K. Vogt, “Akt-mediated regulation of NFkappaB and

the essentialness of NFkappaB for the oncogenicity of PI3K and Akt,” Int. J.
cancer, vol. 125, no. 12, pp. 2863–2870, Dec. 2009, doi: 10.1002/ijc.24748.

[45] L. P. Kane, M. N. Mollenauer, Z. Xu, C. W. Turck, and A. Weiss, “Akt-

Dependent Phosphorylation Specifically Regulates Cot Induction of NF-κB-
Dependent Transcription,” Mol. Cell. Biol., vol. 22, no. 16, pp. 5962 LP – 5974,
Aug. 2002, doi: 10.1128/MCB.22.16.5962-5974.2002.

[46] M.-C. Weng et al., “Apoptosis induction and AKT/NF-κB inactivation are
associated with regroafenib-inhibited tumor progression in non-small cell lung
cancer in vitro and in vivo,” Biomed. Pharmacother., vol. 116, p. 109032, 2019,
doi: https://doi.org/10.1016/j.biopha.2019.109032.

[47] H. Goto et al., “Antitumor effect of berberine against primary effusion

 lymphoma via inhibition of NF-κB pathway.,” Cancer Sci., vol. 103, no. 4, pp.
775–781, Apr. 2012, doi: 10.1111/j.1349-7006.2012.02212.x.

[48] D. Sengupta et al., “Modulation of adenylate cyclase signaling in association

with MKK3/6 stabilization under combination of SAC and berberine to reduce
HepG2 cell survivability,” Apoptosis, vol. 22, no. 11, pp. 1362–1379, 2017, doi:
10.1007/s10495-017-1407-x.

[49] T. P. Hamsa and G. Kuttan, “Berberine Inhibits Pulmonary Metastasis through

Down-regulation of MMP in Metastatic B16F-10 Melanoma Cells,” Phyther.
Res., vol. 26, no. 4, pp. 568–578, Apr. 2012, doi:
https://doi.org/10.1002/ptr.3586.

[50] B. Chen, J. Liu, T.-T. Ho, X. Ding, and Y.-Y. Mo, “ERK-mediated NF-κB
activation through ASIC1 in response to acidosis,” Oncogenesis, vol. 5, no. 12,
pp. e279–e279, 2016, doi: 10.1038/oncsis.2016.81.

[51] Z.-Z. Chen, “Berberine Induced Apoptosis of Human Osteosarcoma Cells by

Inhibiting Phosphoinositide 3 Kinase/Protein Kinase B (PI3K/Akt) Signal
Pathway Activation,” Iran. J. Public Health, vol. 45, no. 5, pp. 578–585, May
2016, [Online]. Available: https://pubmed.ncbi.nlm.nih.gov/27398330.

[52] J. Yue and J. M. López, “Understanding MAPK Signaling Pathways in

Apoptosis,” Int. J. Mol. Sci., vol. 21, no. 7, p. 2346, Mar. 2020, doi:
10.3390/ijms21072346.

[53] N. Yip K.Y. and W. Ho S., “Berberine induces apoptosis via the mitochondrial
pathway in liver cancer cells,” Oncol Rep, vol. 30, no. 3, pp. 1107–1112, 2013,
doi: 10.3892/or.2013.2543.

[54] L. Li et al., “Berberine Suppressed Tumor Growth through Regulating Fatty

Acid Metabolism and Triggering Cell Apoptosis via Targeting FABPs,”
Evidence-Based Complement. Altern. Med., vol. 2020, p. 6195050, 2020, doi:
10.1155/2020/6195050.

[55] B. I. Carr et al., “Fluoro-Sorafenib (Regorafenib) effects on hepatoma cells:

growth inhibition, quiescence, and recovery,” J. Cell. Physiol., vol. 228, no. 2,
pp. 292–297, Feb. 2013, doi: 10.1002/jcp.24148.

[56] R. D’Alessandro et al., “Reversibility of regorafenib effects in hepatocellular

carcinoma cells,” Cancer Chemother. Pharmacol., vol. 72, no. 4, pp. 869–877,
Oct. 2013, doi: 10.1007/s00280-013-2269-8.

[57] Y. Huang et al., “Berberine, a natural plant alkaloid, synergistically sensitizes

human liver cancer cells to sorafenib,” Oncol Rep, vol. 40, no. 3, pp. 1525–
1532, 2018, doi: 10.3892/or.2018.6552.

[58] Y. A. Fouad and C. Aanei, “Revisiting the hallmarks of cancer,” Am. J. Cancer
Res., vol. 7, no. 5, pp. 1016–1036, May 2017, [Online]. Available:
https://pubmed.ncbi.nlm.nih.gov/28560055.

[59] A. A. and A. S. Tarnawski, “Critical Role of Hypoxia Sensor - HIF-1α in VEGF

 Gene Activation. Implications for Angiogenesis and Tissue Injury Healing,”
Current Medicinal Chemistry, vol. 19, no. 1. pp. 90–97, 2012, doi:
http://dx.doi.org/10.2174/092986712803413944.

[60] G. N. Masoud and W. Li, “HIF-1α pathway: role, regulation and intervention for
cancer therapy,” Acta Pharm. Sin. B, vol. 5, no. 5, pp. 378–389, Sep. 2015,
doi: 10.1016/j.apsb.2015.05.007.

[61] S. Grugel, G. Finkenzeller, K. Weindel, B. Barleon, and D. Marmé, “Both v-Ha-

Ras and v-Raf Stimulate Expression of the Vascular Endothelial Growth Factor
in NIH 3T3 Cells,” J. Biol. Chem., vol. 270, no. 43, pp. 25915–25919, Oct.
1995, doi: 10.1074/jbc.270.43.25915.

[62] C. Mirantes et al., “Effects of the multikinase inhibitors Sorafenib and

Regorafenib in PTEN deficient neoplasias,” Eur. J. Cancer, vol. 63, pp. 74–87,
2016, doi: https://doi.org/10.1016/j.ejca.2016.04.019.

[63] L. Mao et al., “Berberine decelerates glucose metabolism via suppression of

mTOR-dependent HIF-1α protein synthesis in colon cancer cells,” Oncol Rep,
vol. 39, no. 5, pp. 2436–2442, 2018, doi: 10.3892/or.2018.6318.

[64] F. I. Montalto and F. De Amicis, “Cyclin D1 in Cancer: A Molecular Connection

for Cell Cycle Control, Adhesion and Invasion in Tumor and Stroma.,” Cells,
vol. 9, no. 12, Dec. 2020, doi: 10.3390/cells9122648.

[65] N. Xu, Y. Lao, Y. Zhang, and D. A. Gillespie, “Akt: A Double-Edged Sword in

Cell Proliferation and Genome Stability,” J. Oncol., vol. 2012, p. 951724, 2012,
doi: 10.1155/2012/951724.

[66] Y. Chen, X. Liu, H. Wang, S. Liu, N. Hu, and X. Li, “Akt Regulated
Phosphorylation of GSK-3β/Cyclin D1, p21 and p27 Contributes to Cell
Proliferation Through Cell Cycle Progression From G1 to S/G2M Phase in
Low-Dose Arsenite Exposed HaCat Cells,” Front. Pharmacol., vol. 10, p. 1176,
2019, doi: 10.3389/fphar.2019.01176.

[67] S. Papa, C. Bubici, F. Zazzeroni, and G. Franzoso, “Mechanisms of liver

disease: cross-talk between the NF-κB and JNK pathways,” vol. 390, no. 10,
pp. 965–976, 2009, doi: doi:10.1515/BC.2009.111.

[68] N. Wang et al., “Berberine Suppresses Cyclin D1 Expression through

Proteasomal Degradation in Human Hepatoma Cells.,” Int. J. Mol. Sci., vol.
17, no. 11, Nov. 2016, doi: 10.3390/ijms17111899.

[69] G. Digiacomo et al., “Simultaneous Combination of the CDK4/6 Inhibitor

Palbociclib With Regorafenib Induces Enhanced Anti-tumor Effects in
Hepatocarcinoma Cell Lines,” Front. Oncol., vol. 10, p. 1880, 2020, doi:
10.3389/fonc.2020.563249.

[70] W.-T. Tai et al., “STAT3 mediates regorafenib-induced apoptosis in

hepatocellular carcinoma.,” Clin. cancer Res. an Off. J. Am. Assoc. Cancer
Res., vol. 20, no. 22, pp. 5768–5776, Nov. 2014, doi: 10.1158/1078-
 0432.CCR-14-0725.

[71] P.-J. Pan, Y.-C. Liu, and F.-T. Hsu, “Protein Kinase B and Extracellular Signal-
Regulated Kinase Inactivation is Associated with Regorafenib-Induced
Inhibition of Osteosarcoma Progression In Vitro and In Vivo.,” J. Clin. Med.,
vol. 8, no. 6, Jun. 2019, doi: 10.3390/jcm8060900.

[72] T. Copetti, C. Bertoli, E. Dalla, F. Demarchi, and C. Schneider, “p65/RelA

Modulates BECN1 Transcription and Autophagy,” Mol. Cell. Biol., vol. 29, no.
10, pp. 2594 LP – 2608, May 2009, doi: 10.1128/MCB.01396-08.

[73] H.-D. Xu and Z.-H. Qin, “Beclin 1, Bcl-2 and Autophagy BT - Autophagy:
Biology and Diseases: Basic Science,” Z.-H. Qin, Ed. Singapore: Springer
Singapore, 2019, pp. 109–126.

[74] Y. Xie, R. Kang, and D. Tang, “Chapter 2 - Role of the Beclin 1 Network in the
Cross-Regulation Between Autophagy and Apoptosis,” M. A. B. T.-A. C. Hayat
Other Pathologies, Inflammation, Immunity, Infection, and Aging, Ed. San
Diego: Academic Press, 2016, pp. 75–88.

[75] M. M. Lipinski, “Towards the global understanding of the autophagy regulatory

network,” Autophagy, vol. 6, no. 8, pp. 1218–1220, Nov. 2010, doi:
10.4161/auto.6.8.13772.

[76] H. Zhang, Z. Chen, R. N. Miranda, L. J. Medeiros, and N. McCarty, “TG2 and

NF-κB Signaling Coordinates the Survival of Mantle Cell Lymphoma Cells via
IL6-Mediated Autophagy,” Cancer Res., vol. 76, no. 21, pp. 6410 LP – 6423,
Nov. 2016, doi: 10.1158/0008-5472.CAN-16-0595.

[77] W. C. Comb, P. Cogswell, R. Sitcheran, and A. S. Baldwin, “IKK-dependent,

NF-κB-independent control of autophagic gene expression.,” Oncogene, vol.
30, no. 14, pp. 1727–1732, Apr. 2011, doi: 10.1038/onc.2010.553.

[78] L. Chen, D. Liu, Y. Zhang, H. Zhang, and H. Cheng, “The autophagy molecule
Beclin 1 maintains persistent activity of NF-κB and Stat3 in HTLV-1-
transformed T lymphocytes.,” Biochem. Biophys. Res. Commun., vol. 465, no.
4, pp. 739–745, Oct. 2015, doi: 10.1016/j.bbrc.2015.08.070.

[79] S. Yoon et al., “NF-κB and STAT3 cooperatively induce IL6 in starved cancer
cells.,” Oncogene, vol. 31, no. 29, pp. 3467–3481, Jul. 2012, doi:
10.1038/onc.2011.517.

[80] N. Wang et al., “Berberine induces autophagic cell death and mitochondrial
apoptosis in liver cancer cells: The cellular mechanism,” J. Cell. Biochem., vol.
111, no. 6, pp. 1426–1436, Dec. 2010, doi: https://doi.org/10.1002/jcb.22869.

[81] R. Yu, Z. Zhang, B. Wang, H. Jiang, L. Cheng, and L. Shen, “Berberine-

induced apoptotic and autophagic death of HepG2 cells requires AMPK
activation,” Cancer Cell Int., vol. 14, no. 1, p. 49, 2014, doi: 10.1186/1475-
2867-14-49.

[82] Y. Xu, J. Yuan, and M. M. Lipinski, “Live imaging and single-cell analysis
 reveal differential dynamics of autophagy and apoptosis,” Autophagy, vol. 9,
no. 9, pp. 1418–1430, Sep. 2013, doi: 10.4161/auto.25080.

[83] P. Bhat, J. Kriel, B. Shubha Priya, Basappa, N. S. Shivananju, and B. Loos,

“Modulating autophagy in cancer therapy: Advancements and challenges for
cancer cell death sensitization,” Biochem. Pharmacol., vol. 147, pp. 170–182,
2018, doi: https://doi.org/10.1016/j.bcp.2017.11.021.

[84] B.-S. Xie et al., “Autophagy inhibition enhances etoposide-induced cell death
in human hepatoma G2 cells.,” Int. J. Mol. Med., vol. 27, no. 4, pp. 599–606,
Apr. 2011, doi: 10.3892/ijmm.2011.607.

[85] J. S. Carew et al., “Autophagy inhibition enhances vorinostat-induced

apoptosis via ubiquitinated protein accumulation.,” J. Cell. Mol. Med., vol. 14,
no. 10, pp. 2448–2459, Oct. 2010, doi: 10.1111/j.1582-4934.2009.00832.x.

[86] S. Shimizu et al., “Inhibition of autophagy potentiates the antitumor effect of

the multikinase inhibitor sorafenib in hepatocellular carcinoma,” Int. J. Cancer,
vol. 131, no. 3, pp. 548–557, Aug. 2012, doi: https://doi.org/10.1002/ijc.26374.

[87] H. Yi et al., “Inhibition of autophagy enhances the targeted therapeutic effect of

sorafenib in thyroid cancer,” Oncol Rep, vol. 39, no. 2, pp. 711–720, 2018, doi:
10.3892/or.2017.6118.