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Berberine as a Potential Anticancer Agent: A Comprehensive Review

Article in Molecules · December 2021


DOI: 10.3390/molecules26237368

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molecules
Review
Berberine as a Potential Anticancer Agent:
A Comprehensive Review
Abdur Rauf 1, * , Tareq Abu-Izneid 2 , Anees Ahmed Khalil 3 , Muhammad Imran 3 , Zafar Ali Shah 1 ,
Talha Bin Emran 4 , Saikat Mitra 5 , Zidan Khan 6 , Fahad A. Alhumaydhi 7 , Abdullah S. M. Aljohani 8 ,
Ishaq Khan 9 , Md. Mominur Rahman 10 , Philippe Jeandet 11, * and Tanweer Aslam Gondal 12

1 Department of Chemistry, University of Swabi, Anbar 23561, Pakistan; zafarali@uoswabi.edu.pk


2 Pharmaceutical Sciences Program, College of Pharmacy, Al Ain University, Al Ain 64141,
United Arab Emirates; tizneid@gmail.com
3 University Institute of Diet and Nutritional Sciences, Faculty of Allied Health Sciences, The University
of Lahore, Lahore 54000, Pakistan; aneesahmedkhalil@gmail.com (A.A.K.); mic_1661@yahoo.com (M.I.)
4 Department of Pharmacy, BGC Trust University Bangladesh, Chittagong 4381, Bangladesh;
talhabmb@bgctub.ac.bd
5 Department of Pharmacy, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh;
saikatmitradu@gmail.com
6 Department of Pharmacy, International Islamic University Chittagong, Chittagong 4318, Bangladesh;
zidankhan9090@gmail.com
7 Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University,
 Buraydah 52571, Saudi Arabia; f.alhumaydhi@qu.edu.sa
 8 Department of Veterinary Medicine, College of Agriculture and Veterinary Medicine, Qassim University,
Citation: Rauf, A.; Abu-Izneid, T.; Buraydah 52571, Saudi Arabia; jhny@qu.edu.sa
9 Institute of Basic Medical Sciences, Khyber Medical University, Peshawar 25100, Pakistan;
Khalil, A.A.; Imran, M.; Shah, Z.A.;
Emran, T.B.; Mitra, S.; Khan, Z.;
ishaqkhan.ibms@kmu.edu.pk
10 Department of Pharmacy, Faculty of Allied Health Sciences, Daffodil International University,
Alhumaydhi, F.A.; Aljohani, A.S.M.;
Dhaka 1207, Bangladesh; mominur.ph@gmail.com
et al. Berberine as a Potential 11 University of Reims Champagne-Ardenne, Research Unit, Induced Resistance and Plant Bioprotection,
Anticancer Agent: A Comprehensive
EA 4707, USC INRAe 1488, SFR Condorcet FR CNRS 3417, Faculty of Sciences, P.O. Box 1039, CEDEX 2,
Review. Molecules 2021, 26, 7368. 51687 Reims, France
https://doi.org/10.3390/ 12 School of Exercise and Nutrition, Faculty of Health, Deakin University, Burwood, VIC 3125, Australia;
molecules26237368 tgondal@deakin.edu.au
* Correspondence: mashaljcs@yahoo.com (A.R.); philippe.jeandet@univ-reims.fr (P.J.)
Academic Editors: Enrique Barrajon,
Vicente Micol and María Abstract: Berberine (BBR), a potential bioactive agent, has remarkable health benefits. A substantial
Herranz-López amount of research has been conducted to date to establish the anticancer potential of BBR. The
present review consolidates salient information concerning the promising anticancer activity of this
Received: 13 November 2021
compound. The therapeutic efficacy of BBR has been reported in several studies regarding colon,
Accepted: 2 December 2021
breast, pancreatic, liver, oral, bone, cutaneous, prostate, intestine, and thyroid cancers. BBR prevents
Published: 4 December 2021
cancer cell proliferation by inducing apoptosis and controlling the cell cycle as well as autophagy.
BBR also hinders tumor cell invasion and metastasis by down-regulating metastasis-related proteins.
Publisher’s Note: MDPI stays neutral
Moreover, BBR is also beneficial in the early stages of cancer development by lowering epithelial–
with regard to jurisdictional claims in
published maps and institutional affil-
mesenchymal transition protein expression. Despite its significance as a potentially promising drug
iations. candidate, there are currently no pure berberine preparations approved to treat specific ailments.
Hence, this review highlights our current comprehensive knowledge of sources, extraction methods,
pharmacokinetic, and pharmacodynamic profiles of berberine, as well as the proposed mechanisms
of action associated with its anticancer potential. The information presented here will help provide a
Copyright: © 2021 by the authors.
baseline for researchers, scientists, and drug developers regarding the use of berberine as a promising
Licensee MDPI, Basel, Switzerland. candidate in treating different types of cancers.
This article is an open access article
distributed under the terms and Keywords: berberine; alkaloids; pharmacokinetic study; cancer preventive agents; cancer
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Molecules 2021, 26, 7368. https://doi.org/10.3390/molecules26237368 https://www.mdpi.com/journal/molecules


Molecules 2021, 26, 7368 2 of 19

1. Introduction
Cancer is a disease that has become a significant public health and socio-economic
concern worldwide. Hence, it seems urgent to develop strategies for the prevention and
treatment of cancer [1]. Various types of cancers display resistance to chemo, radio, and
hormonal therapies. Owing to these limitations, there is a dire need to develop effec-
tive, readily available, and safe anticancer therapies. Consequently, researchers are now
more focused on exploring natural plant components as potential anticancer agents [2].
Plants produce numerous distinct natural products—secondary metabolites—such as ter-
penoids, phenolics, and alkaloids. In matrices of higher plants, phenolics and terpenoids
are more abundantly present than alkaloids [3]. Among alkaloids, isoquinoline alkaloids
are known as natural plant products that have demonstrated a considerable impact in
drug discovery. Isoquinoline alkaloids are predominantly present in diverse plant families
such as Berberidaceace, Cactaceae, Rutaceae, Fumariaceae, Papaveraceace, Magnoliaeace,
Menispermaceae, Amaryllidaceae, and Ranunculaceae. These alkaloids have remarkable bi-
ological and pharmacological properties such as antifungal, anti-inflammatory, antioxidant,
anticancer, antihypercholesterolemic, antidiabetic, and antimicrobial [4–6].
Berberine (BBR) is a benzyl tetra isoquinoline alkaloid (2,3-methylenedioxy-9,10-dimethoxy
protoberberine chloride, C20 H18 NO4 + ) (Figure 1) with a molar mass of 336.36122 g/mol. It
is a well-known phytochemical compound extracted from the roots of various plants such
as Berberis vulgaris, B. aristotle, B. aquifolium, Hydrastus canadensis, Pellodendron chenins, and
Coptis rhizomes [7,8]. It is a crystal yellow-colored isoquinoline alkaloid traditionally used
in Chinese and Ayurvedic medicine. Recently, scientists have reported that Berberine pos-
sesses broad-spectrum therapeutic potential due to its action against various ailments such
Molecules 2021, 26, x FOR PEER REVIEW 4 of 22
as diabetes, hypertension, depression, obesity, inflammation, and cancer [9–13]. Berberine
sulfate and hydrochloride have also been considered efficient herbal treatments. Scientists
have reported intestinal tract helps
berberine asconvert berberine todrug
a promising its easily absorbable form,
candidate dihydroberberine,
in treating cancer [14] and various
which displays a 5-fold higher intestinal absorption rate compared with its parent mole-
diseases such culeas[36].
diabetes, Alzheimer’s.
Following administration of BBRItinisrats
a and
hydrophilic compound
humans, the presence of the BBRhaving low bioavail-
ability whenmetabolitesadministratedM1, M2, M3, and M4 therefore,
orally; was detected invarious
bile, urine, nanotechnology-based
and feces, as well as BBR strategies are
sulfate and glucuronide conjugates [37]. The pharmacokinetic profile of BBR and its me-
in practice totabolites,elevate berberine
which bioavailability.
was extensively studied both in animal Furthermore,
models [38] and coadministration
in humans [39], with certain
drugs resultsdemonstrated
in increased analogies between the two models, mainly regarding the low oral bioavail-
absorption of berberine. Additionally, for decades it has served
ability of BBR, thus requiring relatively high dosages for clinical practice (0.5–1 g/month).
as a chemical Chenmarker in assessing
et al. studied the quality
the BBR pharmacokinetic profileofin various
rabbits after prescriptions
intravenous admin- in clinical use [15].
istration of 2 mg/kg BBR sulfate, obtaining the following kinetic parameters; t
Therefore, this review
1.18 min, t
1/2(β)
summarizes the pharmacokinetic profile
1/2(α)
of: berberine
: 5.28 ± 1.00 h, total plasma clearance (CL): 5.46 ± 1.62 L/h, elimination rate
2.32 ±
and presents
an in-depth constant overview10 of ±its
(K ): 1.75 1.17anticancer
−1 h , and an areaperspectives.
under the concentration-time curve (AUC): 0.84
± 0.27 μg h/mL [40].

Figure 1. Chemical structures of berberine and its primary metabolites.


Figure 1. Chemical structures of berberine and its primary metabolites.
Spinozzi et al. have reported that after a single oral intake of BBR chloride in healthy
subjects (500 mg), plasmatic BBR, M3, and M4 levels (Figure 1) were very low (0.07 ± 0.01,
0.14 ± 0.01, and 0.13 ± 0.02 nM, respectively) displaying a similar pharmacokinetic profile;
a plateau was reached after one hour for BBR and M3 and after 2 h for M4, persisting for
up to 24 h [41]. In contrast, the plasma concentration of M1 reached 10-fold higher levels
after 4 h, that is, 1.4 ± 0.3 nM, slowly decreasing to a concentration of 0.15 ± 0.02 nM after
24 h. The same authors reported that after a chronic administration of 15 mg/kg body
Molecules 2021, 26, 7368 3 of 19

2. Sources and Extraction Techniques


Various parts (bark, stem, root, and rhizome) of plants such as goldenseal (Hydrastis
canadesis), goldenthread (Coptis chinesis), barberry (Berberis vulgaris), Oregon grape (Berberis
aquifolium), and tree turmeric (Berberis aristata) are known to contain active biomolecules
such as berberine. Further, berberine has also been extracted and isolated from diverse plant
genera and families, including Tinospora (Menispermaceae), Annickia (Annonaceae), Xan-
thorhiza (Ranunculaceae), Sinopodophyllum (Berberidaceae), Evodia (Rutaceae), Coelocline
(Annonaceae), Argemone (Papaveraceae), Rollinia (Annonaceae), Caulophyllum (Berberi-
daceae), Zanthoxyllum (Rutaceae), Xylopia (Annonaceae), Bocconia (Papaveraceae), and
others. Among these plants, berberine is abundantly present in several species of barberry
and goldenseal that are native to America and Asia [16–18].
As discussed above, berberine is an alkaloid predominantly present in the matrices
of various plant species, and a variety of solvents are used for its isolation. Principally,
extraction methods used to isolate berberine depend on interconversion reactions among
the protoberberine salt and the base itself. Apart from extraction methodologies, conversion
of protoberberine salts to their specific bases is performed, and the resulting bases are
further extracted using different organic solvents [19,20]. As berberine is a photo- and
thermo-sensitive compound, both light and heat are considered as main challenging factors
during its extraction. However, various conventional extraction methods are widely used,
such as soxhlet, percolation, maceration, and continuous hot extraction, using different
solvents (chloroform, ethanol, and methanol). In these conventional methods, exposure to
light and heat results in the degradation of berberine, thereby reducing berberine recovery
from plant matrices [21]. Currently, research is focused on employing novel and innovative
extraction techniques (supercritical fluid or pressurized liquid extractions, ultrasonication,
microwave-assisted extraction, and ultrahigh pressure extraction) due to their enhanced
extraction efficiency, reduced extraction time, and minimal detrimental effects [22]. Choices
of the solvent and the type of extraction technique are considered critical steps in both the
extraction and the isolation of berberine. Table 1 gives a brief overview regarding berberine
extraction using different techniques.

Table 1. Overview of various techniques for berberine extraction.

Source Plant Part Extraction Method(s) References


Microwave-assisted subcritical
Berberis aristata Roots [23]
water extraction
Coscinium fenestratum Stems Sonication [24]
Berberis lyceum Roots Soxhlet extraction [25]
Coscinium fenestratum Stems Hot and cold extraction [26]
Berberis aristata Stem bark Hot extraction [27]
Stems, leaves, and Maceration and pulsed electric
Berberis integerrima [28]
fruits field assisted extraction
Coptis chinensis Rhizome Supercritical fluid extraction [29]
Ultrahigh pressure extraction,
Phellodendri amurensis cortex Barks ultrasonic extraction, soxhlet [30]
extraction, heat reflux extraction
Berberis tinctoria Stem bark Hot extraction [27]
Stems, leaves and Maceration and pulsed electric
Berberis thunbergii [28]
fruits field assisted extraction
Phellodendri amurensis cortex Barks Ultrasound-assisted extraction [31]
Pressurized hot water extraction,
Hydrastis canadensis Roots [32]
reflux extraction, ultrasonication
Microwave-assisted extraction,
Tinospora cordifolia Stems [33]
soxhlet extraction, maceration
Mahonia manipurensis Stem bark Cold extraction [34]
Molecules 2021, 26, 7368 4 of 19

3. Pharmacokinetic Profile of BBR


In humans and mice, the primary metabolites of BBR are berberrubin (M1), thalifen-
dine (M2), demethyleneberberine (M3), and jatrorrhizine (M4), as other alkaloids contained
in the extracts of H. candidiasis (such as hydrastine) (Figure 1) [35,36]. The bacterial mi-
croflora of the intestine plays an important role in the enterohepatic circulation of BBR and
its regulated metabolites. Recent reports have shown that the microbiota of a healthy in-
testinal tract helps convert berberine to its easily absorbable form, dihydroberberine, which
displays a 5-fold higher intestinal absorption rate compared with its parent molecule [36].
Following administration of BBR in rats and humans, the presence of the BBR metabolites
M1, M2, M3, and M4 was detected in bile, urine, and feces, as well as BBR sulfate and
glucuronide conjugates [37]. The pharmacokinetic profile of BBR and its metabolites, which
was extensively studied both in animal models [38] and in humans [39], demonstrated
analogies between the two models, mainly regarding the low oral bioavailability of BBR,
thus requiring relatively high dosages for clinical practice (0.5–1 g/month). Chen et al.
studied the BBR pharmacokinetic profile in rabbits after intravenous administration of
2 mg/kg BBR sulfate, obtaining the following kinetic parameters; t1/2(α) : 2.32 ± 1.18 min,
t1/2(β) : 5.28 ± 1.00 h, total plasma clearance (CL): 5.46 ± 1.62 L/h, elimination rate con-
stant (K10 ): 1.75 ± 1.17 h−1 , and an area under the concentration-time curve (AUC):
0.84 ± 0.27 µg h/mL [40].
Spinozzi et al. have reported that after a single oral intake of BBR chloride in healthy
subjects (500 mg), plasmatic BBR, M3, and M4 levels (Figure 1) were very low (0.07 ± 0.01,
0.14 ± 0.01, and 0.13 ± 0.02 nM, respectively) displaying a similar pharmacokinetic profile;
a plateau was reached after one hour for BBR and M3 and after 2 h for M4, persisting
for up to 24 h [41]. In contrast, the plasma concentration of M1 reached 10-fold higher
levels after 4 h, that is, 1.4 ± 0.3 nM, slowly decreasing to a concentration of 0.15 ± 0.02 nM
after 24 h. The same authors reported that after a chronic administration of 15 mg/kg
body weight/day of BBR for three months, patients with hypercholesterolemia showed
plasmatic bioaccumulation of BBR and its primary metabolites. Maximum steady-state
concentrations were 4.0 ± 2.0, 6.7 ± 3.0, 1.7 ± 0.3, and 5.6 ± 2.0 nM for BBR, M1, M3, and
M4, respectively. Even so, M1 was the most abundant compound present in the plasma [41].
Despite the low plasmatic concentration of BBR and low bioavailability, its metabolites
retained a higher concentration in the plasma, behaving as pharmacologically active forms
of BBR [42,43]. Moreover, after oral administration, BBR is rapidly distributed in the body
with maximum concentrations in the liver, followed by kidney, muscles, lung, brain, heart,
pancreas, and fat [44].

4. Anticancer Perspectives
4.1. Breast Cancer
Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype. Berberine
was cytotoxic against all treated TNBC cell lines such as MDA-MB-231, MDA-MB-468,
HCC1937, HCC70, HCC38, BT-20, HCC1143, and BT-549. Among all these experimented
cell lines, the most sensitive ones were HCC70 (IC50 = 0.19 µM), BT-20 (IC50 = 0.23 µM), and
MDA-MB-468 (IC50 = 0.48 µM) [45]. Using flow cytometry techniques, BBR at 0.5 and 1 µM
for 120 and 144 h not only induced cell cycle arrest at first growth (G1) and second-growth
(G2)/medium phases, but it also triggered significant apoptosis [45]. Interestingly, although
BBR was cytotoxic to TNBC cells, it did not affect the viability of normal human breast
cells (MCF10) cultured in a 3D Matrigel model 15. These results suggest that berberine
may be a suitable potential candidate for the development of a TNBC drug. Berberine
addition at a dose of 1 µM to MDA-MB-468 cells induced a significant increase in the G1
phase population with a decrease in the S and G2/M phases [46].
BBR reduced the expression of the proliferating cell nuclear antigen (PCNA) protein
and cyclin D1 in MDA-MB-468 cell cultures to block their progression into the G1 phase
of the cell cycle. Likewise, application of BBR to MDA-MB-468 cells at a dose of 6 and
12 µM for 48 h caused a cell cycle arrest in the first growth (G1) phase with a decrease
Molecules 2021, 26, 7368 5 of 19

in the expression of cyclin D1 depending on the dose [46]. Zhao and Zhang recently
investigated the role of berberine regarding the behavior of the MDA-MB-231 malignant
breast tumor cell line. Namely, BBR reduced cell migration ability, provoked inhibition
of phosphorylation, decreased overexpression of the tumor necrosis factor α (TNF-α)
and Interleukin 6 (IL-6), and induced suppression of the nuclear factor kappa light chain
enhancer of activated β cells (NF-Kβ) [47].
On the other hand, autophagy is a conservative mechanism for maintaining cellular
homeostasis by clearing misfolded proteins and damaged organelles [48]. In cancer therapy,
autophagy is seen as a double-edged sword because it can prevent early tumorigenesis
and protect cancer cells later in life [49]. Thus, the combination of autophagy inhibitors
and chemotherapy is expected as a promising cancer treatment strategy, and multiple
autophagy inhibitors are already in the preclinical stage [50].
In MCF-7 breast cancer cells and the doxorubicin-resistant (ADR) cells MCF-7 (MCF-7/
ADR), BBR was recently identified as an autophagy suppressor, inhibiting the formation of
autophagosomes in MCF-7/ADR cells [51]. Berberine treatment blocked the accumulation
of the LC3II protein, which is associated with autophagy, leading to accumulation of the
signaling adaptor p62 protein, decreasing cell proliferation, and reversing doxorubicin re-
sistance [52]. Mechanically, BBR inhibits autophagy by modulating the PTEN/Akt/mTOR
signaling pathway. It also regulates the mitogen-activated protein kinase and the Wing-
less/Integrated (Wnt)/β-catenin signaling pathways in breast cancer cells [53] while sup-
pressing chemotherapy resistance through autophagy regulation [46,54]. BBR as a potent
anticancer agent significantly reduces cell viability, inhibits colony formation, cell migration,
and decreases the secretion of proinflammatory cytokines (IL-1α, IL-6, TNF-α, IL-1β) [55].
BBR also increases the release of Lactic Acid Dehydrogenase (LDH) in the MDA epithelial
human breast cancer cell line (MDA-cells) and downregulates the purinoceptor 7 (P2 × 7)
associated with speck apoptosis, procaspase-1, and caspase-1 p20, domain recruitment
(ASC), IL-1β proteins, interleukin-18 (IL-18), the mRNA expression of caspase-1 and ASC
in the NOD-, and LRR- and the pyrin domain-containing protein 3 (NLRP3) inflammasome
cascade [55]. Proposed mechanisms regarding the breast anticancer properties of berberine
are presented in Table 2.

4.2. Colon Cancer


BBR treatment suppresses the viability of colorectal cancer cells by increasing their
apoptosis level. The long noncoding RNA cancer susceptibility candidate 2 (CASC2) is
activated in cells treated with BBR, and knockdown of the RNA CASC2 reverses BBR-
induced apoptosis [56]. In addition, the antiapoptotic β-cell lymphoma-2 (Bcl-2) gene and
CASC2 were inhibited by treatment with berberine causing proapoptotic effects. Moreover,
CASC2 lncRNA binds to the Au-rich element-binding factor 1 (AUF1), which blocks the
binding of AUF1 to Bcl-2 mRNA, thereby inactivating Bcl-2 translation [56]. There are
many antitumor mechanisms induced by BBR in human colorectal cancer cells, such
as suppression of cell viability, induction of cell apoptosis, and upregulation of CASC2
lncRNA [56,57]. Berberine also modulates the expression of the micro-RNA-429 (MIR-429)
in colorectal cancer [58]. The role of BBR in the colorectal cancer stem cells (CRC) was
further explored by Liu et al. [58], who showed that this compound inhibits the invasion
and metastasis of CRC cells via the prostaglandin–endoperoxide synthase 2/prostaglandin
E2, mediated by the Janus kinase 2 pathway [58]. BBR inhibits the viability of CRC cell lines
and promotes cell apoptosis in a dose-dependent manner. Moreover, RNA sequencing has
shown that several lncRNAs may be important regulators of the BBR-dependent pathway.
MiR-21 is involved in cell proliferation, invasion, invasion of blood vessels, and metastasis
of many types of cancers [59]. Berberine suppresses the viability of colon cancer cells and
regulates the three-gene network microRNA (miR)-21-integrin β4 (ITGβ4)—programmed
cell death 4 (PDCD4) [60]. It was demonstrated that BBR treatment suppresses the viability
of colon cancer cells, induces apoptosis, and activates caspase-3 activity in the human
colon cancer cell line HCT116 [60]. BBR inhibits the miR-21 expression and stimulates the
Molecules 2021, 26, 7368 6 of 19

expression of PDCD4 proteins in the HCT116 cell line. Overexpression of miR-21 reduces
the anticancer effects of BBR on cell viability, apoptosis rate, and caspase-3 activity of the
HCT116 cell line [60,61]. Table 2 provides an overview of berberine action against various
colon cancer cell lines and the proposed anticancer mechanisms.

4.3. Pancreatic Cancer


Berberine (0.3–6 µM) inhibits DNA synthesis and proliferation of pancreatic ductal
adenocarcinoma (PDAC) cells and retards the development of their cell cycle in G1. BBR
treatment also reduces by 70% the growth of MiaPaCa-2 cells when implanted into the
flanks of nu/nu mice [62]. BBR lowers mitochondrial membrane potential and intracellular
ATP levels and induces potent AMPK activation, as evidenced by phosphorylation of the
AMPK α subunit at Thr172 and acetyl CoA carboxylase (ACC) at Ser79. In addition, BBR
inhibits, in a dose-dependent manner, mTORC1 (phosphorylation of S6K at Thr389 and S6
at Ser240/244) and ERK activation in PDAC cells stimulated with insulin and neurotensin or
fetal bovine serum [62]. Knockdown of the expression of the catalytic subunits α1 and α2 of
AMPK reverses the inhibitory effect caused by the treatment with low concentrations of BBR
on mTORC1, ERK, and DNA synthesis in PDAC cells. However, at higher concentrations
(3 µM), BBR inhibits mitogenic signaling (mTORC1 and ERK) and DNA synthesis through
an AMPK-independent mechanism [62]. Similar results were obtained with metformin
used at doses that produced either a moderate or significant decrease in intracellular ATP
levels, almost identical to the decrease in ATP levels observed in response to BBR [62]. One
can hypothesize that BBR and metformin inhibit mitogenic signaling in PDAC cells via
dose-dependent AMPK-dependent and independent pathways [63].
G-protein coupled receptors (GPCRs), and their related agonists are used as au-
tocrine/paracrine growth factors for multiple solid tumors [64,65]. It has been shown
that pancreatic cancer cell lines express multiple GPCRs [66] and various GPCR agonists,
including neurotensin, angiotensin II, and bradykinin, which stimulate DNA synthesis
in pancreatic cancer cell lines including PANC-1 and MiaPaca-2 [67]. In the pancreatic
cancer cell lines PANC-1 and MIA-PaCa2, Park et al. [68] identified the anticancer role of
berberine via a variety of pathways such as induction of phase G1. In contrast, induction
of apoptosis was triggered by a mechanism involving the production of reactive oxygen
species (ROS) rather than activation of caspase 3/7. Similarly, in another study, the effects
of berberine and some of the modified berberines (NAX-compounds), metformin, and
chemo-preventive drugs were assessed on four pancreatic adenocarcinoma cell lines (AsPC-
1, BxPC-3, MIA-PaCa-2, and PANC-28). Berberine and modified berberine compounds
enhanced the effects of metformin. In MIA-PaCa-2 cells, restoration of WT-TP53 activity
changed the sensitivity towards metformin and modified BBRs combination compared
with parent cells lacking in WT-TP53. Some modified BBRs helped alter the expression of
key molecules involved in cellular growth. Therefore, the outcomes of that study concluded
that combined treatment with berberines and NAX compounds may help suppress the
proliferation of pancreatic cancer cells [69]. Table 2 highlights the effect of berberine against
various pancreatic cancer cell lines along with its proposed mechanisms of action.
Reportedly, in human pancreatic cancer cells (BxPC-3 cells), BBR has been found to
have an inhibitory action on the cellular growth of cancer cells and mediated caspase-
independent cell death [70]. BBR showed inhibitory effects in pancreatic cancer cells
(PANC-1, AsPC-1, and MIA-PaCa-2) on the expression of Rad51 and the upregulation of
PARP expression compared with control pancreatic cancer cells. The combined influence of
olaparib (PARP inhibitor) and berberine displayed synergistic inhibitory effects on cellular
activity and induced apoptotic conditions in experimented pancreatic cancer cells [71].
Based on a phenotypic assay, berberine showed a notable inhibitory role in pancreatic cancer
cell metastasis and viability. Additionally, berberine treatment significantly damaged the
mitochondria of pancreatic cancer cells and therefore dysregulated their energy metabolism
processes [72]. In pancreatic cancer cells, BBR treatment also influenced citrate metabolism
resulting in blocking of the fatty acid biosynthesis. Finally, Liu et al. [72] have proposed
Molecules 2021, 26, 7368 7 of 19

that BBR inhibits the proliferation of pancreatic cancer cells via the regulation of citrate
metabolism and, therefore, citrate metabolism may be considered a promising target
in drug development for the treatment of pancreatic cancers. Similarly, according to
another study carried out in the pancreatic cancer cells PANC-1, treatment of gemcitabine
(a standard drug) and BBR resulted in the reduction of side-population cells to 6.8 and
5.7%, respectively. Further, in BBR and gemcitabine-treated PANC-1 and MIA-PaCa-2 cells,
all the examined stem cell-associated genes (NOTCH1, NANOG, POU5F1, and SOX2) were
suppressed, except NOTCH1. Hence, the authors believed that the stem cell-associated
genes (NANOG, POU5F1, and SOX2) may serve as promising markers and that BBR can be
considered a potent anticancer agent for the treatment of pancreatic cancers [73].

4.4. Gastric Cancer


Matrix metalloproteinases (MMP) can cleave all extracellular matrix components and
contribute to malignant cell invasion and metastasis. Gastric cancer has been linked to four
matrix metalloproteinases (MMPs) (MMP-1, -2, -7 and -9) [74]. BBR was shown to suppress
human gastric cancer cell growth and migration in a dose-dependent manner. In the
gastric cancer cells SNU-5, BBR induced the production of Reactive Oxygen Species (ROS)
while decreasing the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).
BBR exerted anticancer properties in gastric cancer cells by preventing cell migration by
inhibiting MMP -1, -2, and -9 gene expression [75].
Pandey et al. [76] discovered that BBR impairs gastric cancer cell viability in a dose-
dependent manner by inhibiting the signal transducer and activator of transcription 3
(STAT3) levels and survivin expression. These authors showed that 5-fluorouracil in combi-
nation with BBR increases gastric adenocarcinoma cell death by suppressing survivin and
STAT3 expression [76]. BBR was found to suppress the activation of the epidermal growth
factor receptor (EGFR) in gastric cancer tumors. Research conducted by Wang et al. [77]
evaluated whether BBR could help EGFR tyrosine kinase inhibitors (TKI) function better in
gastric cancer cell lines and xenograft models. They reported that BBR could effectively
improve the activity of targeted standard cancer drugs such as erlotinib and cetuximab
in vitro and in vivo. BBR has been shown to suppress growth and cause apoptosis in
gastric cancer cell lines owing to the inhibition of EGFR signaling, which includes STAT3
phosphorylation [77]. Likewise, in gastric cancer cells (SGC7901 and AGS lines), BBR
treatment suppressed cell proliferation, induced cell cycle arrest, and attenuated invasion
via the down-regulation of C-myc, cyclin-D1, and MMP-3 expressions, respectively [78]. In
reaction to BBR therapy, expression of Bcl-xL and cyclin D1 protein decreased, whereas
cleavage levels of poly-ADP ribose polymerase (PARP) increased significantly [77].
In another study, the effect of BBR has also been examined in gastric cancer cells (SGC-
7901 and BGC-823 lines) that were resistant to cisplatin. Purposely, coadministration of
BBR and cisplatin increased the apoptotic conditions in the experimented cisplatin-resistant
gastric cancer cell lines. Conclusively, it was noticed that BBR sensitized resistant cancer
cells to cisplatin and increased its antigastric cancer properties owing to the inhibition
of PI3K/AKT/mTOR signaling [79]. Berberine-treated gastric cancer cells (BGC-823 and
SGC-7901), which were already resistant towards cisplatin, showed a reduction in cisplatin
resistance due to modulatory effects on the miR-203/Bcl-w apoptotic axis and hence might
increase the chemotherapeutic responses among patients having cisplatin-resistant gastric
cancers [80]. In vitro and in vivo experimentations revealed the inhibitory potential of
BBR in the gastric cancer cell line BGC-823 due to the induction of cytostatic autophagy
through suppression of MAPK/mTOR/p70S6K and Akt signaling pathways [81]. Similarly,
Li et al. [82] proposed the use of berberine hydrochloride as a potential drug candidate for
the treatment of gastric cancer as this compound modulates MAPK-signaling pathways [82]
(Table 2).
Molecules 2021, 26, 7368 8 of 19

4.5. Liver Cancer


BBR inhibits cyclin D1 expression in human hepatoma cells both in vitro and in vivo
in a dose- and time-dependent manner [83]. BBR allows the nuclear cyclin D1 to be released
into the cytoplasm for proteasome degradation by increasing cyclin D1 phosphorylation
at the Thr286 location. To foster cyclin D1 ubiquitin-proteasome-dependent proteolysis,
BBR recruits skp, cullin, and the F-box containing transducing–repeat-containing protein
(SCFβ-TrCP ) complex. Further, BBR blocks the turnover of cyclin D1 when β-TrCP is
knocked out [83]. In hepatocellular carcinoma cells (HCC), over-expression of the Solute
Carrier Family 1 Member 5 (SLC1A5) results in a poor prognosis. On the other hand,
BBR has been reported to inhibit the proliferation of Hep3B and BEL-7404 cells in vitro by
suppressing glutamine uptake and inhibiting SLC1A5; however, the increased activity of
SLC1A5 results in an increase in glutamine uptake and an increase in BBR tolerance. In
addition, BBR inhibits the growth of tumor xenografts and the expression of SLC1A5 and
c-Myc in vivo [84].
BBR can cause cell cycle arrest and display anticancer properties in hepatocellular
carcinoma cells (HCC). G1 step cell cycle arrest was observed in Huh-7 and HepG2 cells
treated with BBR [85]. Moreover, it was found that BBR could inactivate the AKT pathway
resulting in suppression of S-phase kinase-related protein 2 (Skp2) expression while it
increased the expression and the nucleocytoplasmic translocation of the Forkhead box
O3a (FoxO3a). On one side, translocated FoxO3a can directly promote transcription of the
CDKIs p21Cip1 and p27Kip1, thus inhibiting Skp2 expression, both of which contribute to
the upregulation of p21Cip1 and p27Kip1. The cell cycle is thus arrested in the HCC/G1
process [85]. BBR application was found to inhibit cell viability in the hepatocarcinoma
cell lines SNU-182, Hep3B, and HepG2, due to a modulating effect on the expression of
multiple tumorigenesis-related gene proteins [86]. Liver anticancer potential for BBR is
mainly due to the regulation of hepatoma cells via interactions among ESR1, TB52, PTGS2,
CCDN1, and MAPK1 pathways, which act on Hub-nodes in those interlinked pathways.
This is related to immune–inflammatory activities such as induction of apoptotic conditions
and proliferation of hepatic cancer cells [87]. According to a study conducted by Huang
et al. [88], the coadministration of BBR and sorafenib synergistically inhibited the prolifer-
ation of human liver cancer cells (HepG2 and SMM-7721) in a concentration-dependent
manner. Similarly, BBR minimized the cell viability of Bel-7404, HepG2, and H22 cell lines
in a time-and concentration-dependent manner. Additionally, BBR significantly inhibited
the expression of COX-2 (cyclooxygenase-2) and cPLA2 (cytosolic phospholipase) but
increased the arachidonic acid to PGE2 (prostaglandin E2) ratio [89]. Interestingly, BBR was
reported to have a selective inhibitory effect on the proliferation of hepatocellular cancer
cells through induction of apoptotic conditions in AMPK-mediated caspase-dependent
mitochondrial pathways through its action rarely resulted in a cytotoxic impact in normal
cells [90] (Table 2).

4.6. Oral Cancer


BBR caused genomic DNA fragmentation, cell morphology alterations, and nuclear
condensation in a dose-dependent manner in KB oral cancer cells [91]. Apoptosis and
enhanced caspase-3 and -7 activities were also observed. BBR has also been shown to
increase the expression of the FasL death receptor ligand [91]. As a result, the proapoptotic
factors, including caspases-3, 8, and 9, as well as the poly (ADP-ribose) polymerase, were
expressed. BBR also greatly improved the expression of proapoptotic factors such as Bax,
Poor, and Apaf-1, Bcl-2, and Bcl-xL, while antiapoptotic factors were downregulated [91].
The activation of caspase-3 and PARP was blocked by Z-VAD-FMK, a cell-permeable
pan-caspase inhibitor [91].
In athymic nude mice, BBR effectively inhibited tumorigenicity and the development
of the EBV-positive NPC cell line C666-1. Successful inhibition of STAT3 activation in NPC
cells within tumor xenografts grown in nude mice well correlates with the inhibition of
tumorigenic development of NPC cells in vivo. BBR blocked constitutive and IL-6-induced
Molecules 2021, 26, 7368 9 of 19

STAT3 activation [92], which resulted in growth inhibition and apoptosis in NPC cells.
IL-6 was found to be secreted by tumor-associated fibroblasts, and conditioned media
from fibroblasts activated STAT3 in NPC cells [92]. BBR or antibodies to IL-6 and IL-6R
may also inhibit STAT3 activation by regulatory media of tumor-associated fibroblasts [93].
Treatment with BBR impaired the development of the human esophageal squamous cell
carcinoma cell line KYSE-70 and the esophageal adenocarcinoma line SKGT4 in a dose-
and time-dependent manner. The inhibitory function of BBR was more sensitive in KYSE-
70 cells than in SKGT4 cells. The number of cells in the G2/M process (25.94%/5.01%)
was higher in KYSE-70 cells treated with 50 µmol/L BBR for 48 h than in the control
(9.77%/1.28%). At 12 and 24 h after treatment, flow cytometric analysis indicated that BBR
significantly increases the KYSE-70 apoptosis population relative to control cells (0.83%
vs. 43.78%, 12 h) [94]. The apoptosis effect of BBR was higher at 24 h compared to 12 h
(81.86% vs. 43.78% p%, p < 0.01). BBR blocked the phosphorylation of rapamycin and
Akt, the mammalian targets of P70-S6-Kinase, and increased AMP-activated protein kinase
phosphorylation in a prolonged fashion, according to Western blotting [94] (Table 2).

4.7. Bone Cancer


In vitro and in vivo administration of BBR to osteosarcoma cells reduces the expression
of caspase-1 and Interleukin-1 (IL-1) in tumor cells and inhibits tumor cell development. It
was suggested for the first time that BBR inhibits the caspase-1/IL-1 inflammatory signaling
axis, resulting in antiosteosarcoma properties [95]. BBR has a possible genotoxic effect
on human osteosarcoma cells, as determined by DNA fragmentation analysis and flow
cytometry, by dramatically increasing apoptosis in a concentration and time-dependent
manner [96]. In the osteosarcoma U-2 OS cells, BBR and BBR nanoparticles made of heparin
(HP), reduced cell viability, arrested the cell cycle in the G1 phase, and reduced expression
of the mouse 2 min 2 homologs (MDM2) [97]. The PI3K/Akt pathway was activated, rising
Bcl-2 (B-cell lymphoma 2) expression. BBR prevents PI3K/AKT activation resulting in
an increased expression of Bax (Bcl-2-associated X protein) and PARP (Poly (ADP-ribose)
polymerase) and decreased expression of Bcl-2 and caspase-3 [97]. Overall, BBR inhibits
the activation of the PI3K/Akt signaling pathway, which hinders human osteosarcoma
U2OS cell proliferation and induces apoptosis [97]. BBR inhibits human chondrosarcoma
cell migration and invasion by downregulating v3 integrins via the protein kinase C (PKC)
and the proto-oncogene tyrosine-protein kinase, c-Src [98].
BBR (40−160 µmol/L) inhibits cell proliferation and IL-6 secretion in U-266 (human,
peripheral blood, multiple myeloma) cells in a time and dosage-dependent manner. BBR, on
the other hand, decreases miR-21 and Bcl-2 levels and induces ROS formation, G2/M step
arrest, and apoptosis in U266 cells [99]. BBR-induced inhibition of cell proliferation and IL-6
secretion was disrupted by overexpression of miR-21. The activity of NF-κB was reduced
by around 50% in U266 cells treated with BBR (80 µmol/L), followed by a substantial
decrease in miR-21 levels. BBR (80–160 µmol/L) increases Set9 (lysine methyltransferase)
levels by more than two-fold, resulting in methylation of the RelA subunit, which in
turn inhibits NF-κB nuclear translocation and miR-21 transcription. In U266 cells treated
with BBR (80 µmol/L), knocking down Set9 with siRNA resulted in a substantial rise in
NF-κB protein levels and a partial recovery of cell proliferation. BBR prevents multiple
myeloma development by downregulating three miRNA clusters and a significant number
of mRNAs via the TP53, Erb, and MAPK signaling pathways. The mir-99a to 125b cluster
may be a potential therapeutic target for multiple myeloma [100].
IL-6 regulates miR-21 transcription in IL-6-dependent human myeloma cell lines
(HMCL) through signal transducers and activators of transcription 3 (STAT3)-related
mechanisms [101]. Importantly, in the absence of IL-6, the ectopic expression of miR-21 is
necessary to maintain the development of IL-6-dependent MM cells. As expected, the tumor
suppressor programmed cell death 4 (PDCD4) is a miR-21 target. MiR-21 regulates PDCD4
directly, according to luciferase reporter review assays. Signal transducers and transcription
activators 3 will target the miR-21 promoter according to bioinformatics analysis (STAT3);
Molecules 2021, 26, 7368 10 of 19

BBR can inhibit miR-21 transcription in multiple myeloma by downregulating IL-6 through
STAT3 downregulation. Apoptosis, G2 step cell cycle arrest, and colony suppression were
also caused by BBR and seed-targeting anti-miR-21 oligonucleotides in multiple myeloma
cell lines (Table 2). Short interfering RNA depletion of PDCD4 could preserve BBR-induced
cytotoxicity in multiple myeloma cells [102]. The anticancer mechanisms of berberine
Molecules 2021, 26, x FOR PEER REVIEW 11 of 22are
presented in Figure 2.

Figure 2. Anticancer mechanisms of berberine.


Figure 2. Anticancer mechanisms of berberine.
4.8.Cancer
4.8. Cancerof of the
the Glioblastoma
Glioblastoma
BBR-mediated apoptosis blocks the AMPK/mTOR/ULK1 pathway and decreases tu-
BBR-mediated apoptosis blocks the AMPK/mTOR/ULK1 pathway and decreases
mor growth in glioblastoma polymorphic (GBM) cells in vivo [103]. The glioma microen-
tumor growth in glioblastoma polymorphic (GBM) cells in vivo [103]. The glioma microen-
vironment is characterized by inflammation. IL-1 and other neuroinflammatory cytokines
vironment is characterized by inflammation. IL-1 and other neuroinflammatory cytokines
secreted by glioma cells are believed to play a role in tumor initiation and progression
secreted by glioma cells are believed to play a role in tumor initiation and progression [104].
[104]. Inflammatory responses and cancer are linked by certain intrinsic pathways, which
Inflammatory responses and cancer are linked by certain intrinsic pathways, which induce
induce cancer-causing genetic changes, with IL-1 playing a key role in these mechanisms.
cancer-causing genetic changes, with IL-1 playing a key role in these mechanisms. IL-1, for
IL-1, for example, is a downstream effector of Ras activation and NF-κB regulatory gene
example,
activation, is awhich
downstream effector
is necessary of Ras activation
to provide a favorableand NF-κB regulatoryfor
microenvironment gene activation,
tumor for-
which is necessary to provide a favorable microenvironment for tumor
mation [105]. A recent second phase of a clinical trial of a recombinant IL-1R antagonist formation [105].
Afor
recent second phase of a clinical trial of a recombinant IL-1R
multiple myeloma has shown a favorable safety profile and reduced morbidity,antagonist for multiple
myeloma
demonstratinghas shown a favorable
that anti-IL-1 safety
therapy is aprofile
viableand reduced
cancer morbidity,
treatment demonstrating
option [106]. BBR inhib-that
anti-IL-1 therapy is a viable cancer treatment option [106]. BBR inhibits
its the inflammatory cytokine caspase-1 activation through ERK1/2 signaling as well as the inflammatory
cytokine caspase-1
glioma cells’ activation
subsequent through ERK1/2
development signaling
of IL-1 and IL-18. as
BBRwell as glioma
therapy cells’ subsequent
also decreases mo-
development
tility and induces apoptosis in U251 and U87 cells [107]. Furthermore, BBR has the apoptosis
of IL-1 and IL-18. BBR therapy also decreases motility and induces poten-
tial to reverse the mechanism of epithelial–mesenchymal metastasis, which is a sign of
tumor invasion [107].
BBR inhibits tumor development by regulating the differentiation and the role of
stem cells and inducing cell death in neuroblastoma cells. Around the same time, inhibit-
ing the adrenergic signal slows neuroblastoma development and increases cell differenti-
Molecules 2021, 26, 7368 11 of 19

in U251 and U87 cells [107]. Furthermore, BBR has the potential to reverse the mechanism
of epithelial–mesenchymal metastasis, which is a sign of tumor invasion [107].
BBR inhibits tumor development by regulating the differentiation and the role of stem
cells and inducing cell death in neuroblastoma cells. Around the same time, inhibiting
the adrenergic signal slows neuroblastoma development and increases cell differentiation.
Calvani et al. [108] have summarized the potential benefits of BBR in inhibiting tumor
growth and development in different types of cancer, especially neuroblastoma [108], BBR
(6.25–200 µmol/L, 6–48 h) impaired cell viability and proliferation of U87 and U251 human
glioblastoma cell lines in BALB/c nude mice (IC50 of 42 and 32 µmol/L, respectively). BBR
(50 µmol/L) prevented HUVEC cell migration in the transwell assay by 67.50 ± 8.14%
and the Matrigel assay by 73.00 ± 1.12% [109]. In the ectopic xenograft form, BBR
(50 mg/kg) greatly decreased tumor weight (401.2 71.5 mg vs. 860.7 117.1 mg in the vehicle
group) [109]. The hemoglobin content was greatly decreased by BBR (28.81 ± 3.64 µg/mg
vs. 40.84 ± 5.15 µg/mg in the vehicle group, p < 0.001). BBR (50 mg/kg) greatly in-
creased the survival rate of mice in a stereotactic xenograft model. BBR inhibited VEGFR2
and ERK phosphorylation [109] (Table 2).

4.9. Skin Cancer


Various studies have revealed the anticancer role of BBR via inhibition of cell migration
and invasion in different human cancer cells. Likewise, BBR administration (0–2 µM)
resulted in an induction of cellular morphological alterations and decreased the number of
viable cells in human melanoma skin cancer cells (A375.S2 and A375.S2/PLX resistant cells).
Furthermore, BBR suppressed the migration and invasion of the melanoma skin cancer cells
A375.S2. Post 24-h treatments with BBR in A375.S2 cells led to an inhibition of SOS-1, p-AKT,
MMP-1, NF-κB, Ras, p-FAK, and MMP-13 gene expression and an increase in the levels of
PI3K and PKC [110]. BBR was found earlier to suppress the proliferation of skin squamous
carcinoma cells (A431) in a time- and concentration-dependent manner. Moreover, BBR
treatment induced different biochemical changes, such as loss of the membrane potential
of mitochondria, cytochrome-c release into the cytosol, and cleavage of the poly (ADP)
ribose polymerase. Results revealed that BBR induces apoptotic conditions and inhibits
skin squamous carcinoma cells [111].
Similarly, Kou et al. demonstrated that BBR decreases the migration and invasion
of melanoma cells B16 cells and diminishes the expression levels of RARα (retinoic acid
receptor-α), p-AKT, and p-PI3K while upregulating the expression levels of RARβ (retinoic
acid receptor-β) and RARγ (retinoic acid receptor-γ). The authors were of the view that
in mouse melanoma B16 cells, BBR reversed the epithelial to mesenchymal transition and
hence can be used as an effective anticancer agent in treating melanoma via regulation of
the PI3K/Akt pathway [112]. Another group of researchers also studied the combined
effect of berberine with doxorubicin on murine melanoma B16F10 cells both in vitro and
in vivo. The combined treatment revealed strong inhibitory effects on cell growth and
induced cell cycle (G2/M) arrest along with the reduction in Kip1/p27. Further, compared
with the control, combined BBR and doxorubicin treatment caused a reduction in tumor
weight (78%) and volume (85%) in B16F10 xenograft. Therefore, the authors suggested
the usage of BBR and doxorubicin as a potent combination for the inhibition of melanoma
cancer cell growth [113]. In melanoma A375 cells, treatment with BBR was also found to
decrease the metastatic potential of cancer cells due to AMPK activation and inhibition of
the ERK-signaling pathway, while the levels of COX-2 proteins were also reduced [114]
(Table 2).

4.10. Uterus or Endometrium Cancer


Among various gynecological malignancies, endometrial cancer (EC) is recognized as
the third most malignant after breast and cervical cancers [115]. Berberine has been reported
to be an effective natural alkaloid having antiendometrial cancer properties. According
to the in vitro and in vivo studies conducted by Wang and Zhang [116], BBR inhibited
Molecules 2021, 26, 7368 12 of 19

proliferation, migration, and invasion as well as metastasis in endometrial cancers. They


further reported that BBR inhibits cancer cells via COX-2/PGE2-signaling pathways. In
endometrial cancer cells, modulation of COX-2 was achieved as berberine activated the
transcription of miR-101 through AP-1 (activator protein-1). Conclusively, BBR may be
a promising candidate in treating EC as it inhibits cancer cells through miR-101/COX-
2/PGE2-signaling pathways [116]. In EC cells, BBR affects the distribution of the cell cycle
and induces apoptotic conditions via activation of the mitochondrial-caspase pathway.
Furthermore, since BBR engaged the PI3K/Akt pathway, it may be recommended as a
functional ingredient for the prevention and treatment of endometrial cancers [117].

Table 2. Summarized data of berberine effects against various cancers and their proposed mechanisms.

Cancer
Experimental Model (s) Dose Proposed Mechanism (s) References
Type
MDA-MB-231,
MDA-MB-468, HCC1937, Induction of G1 and -G2/M phase cell cycle arrest,
0.2, 0.5 and 1.0 µM [45]
HCC70, HCC38, BT-20, Stimulation of apoptosis in cancer cells
HCC1143 and BT-549
Cell cycle arrest at G1 phase,
MDA-MB-468 6 and 12 µM [46]
Decrease in cyclin D1 expression
Reduction of cell migration,
MDA-MB-231 25 µM/L Phosphorylation inhibition, [47]
Breast
Decrease of TNF-α and IL-6 overexpression
Cancer
MCF-7/ADR 100 µM Inhibition of the formation of autophagosomes [51]
Blocking the accumulation of the LC3II protein,
MCF-7/ADR 100 µM Decrease of cell proliferation, [52]
Reversion of doxorubicin resistance
Reduction of cell viability,
Inhibition of colony formation, cell migration, and
MDA-MB-231 2.5–100 µg/mL [55]
decrease of the secretion of the proinflammatory
cytokines (IL-1α, IL-6, TNF-α, IL-1β)
Upregulation of LncRNA CASC2, Suppression of
HT29, HCT116 0–100 µM [56]
Colon Bcl-2 gene
cancer Induction of apoptosis,
HCT116 1, 10 or 100 µM [60]
Promotion of caspase-3 activity
PANC-1, MiaPaCa-2 0.3–6 µM Inhibition of DNA synthesisCell cycle arrest at G1 [62]
Cell cycle arrest at G1,
PANC-1, MiaPaCa-2 15 µM and 10 µM [68]
Induction of apoptosis
AsPC-1, BxPC-3,
MIA-PaCa-2 and 100, 1000 and 10,000 nM Suppression of the proliferation of cancer cells [69]
PANC-28).
Pancreatic
cancer BxPC-3 10–200 µM Mediation of caspase-independent cell death [70]
PANC-1, MiaPaCa-2, Induction of apoptosis,
5 µM [71]
AsPC-1 Inhibition of PARP and Rad51 expression
Damage of the mitochondria of pancreatic cancer cells,
PANC-1 2.5, 3.75, 5 and 10 µM [72]
Targeting citrate metabolism
PANC-1, MiaPaCa-2 10 µM, 15 µM Downregulation of NANOG, POU5F1, and SOX2 [73]
SNU-5 75 µM Inhibition of MMP-1, -2 and -9 gene expression [75]
AGS 0–50 µM Suppression of survivin and STAT3 expression [76]
SGC7901, MKN45, Downregulation of the expression of Bcl-xL and
15–90 µM [77]
BGC823 cyclin-D1 proteins
Gastric Cell cycle arrest,
cancer SGC7901, AGS 10–80 µM Attenuation of tumor invasion via the down-regulation [78]
of C-myc, cyclin-D1, and MMP-3 expressions
BGC-823, SGC-7901 1–1000 µM Inhibition of PI3K/AKT/mTOR signaling [79]
BGC-823, SGC-7901 10 µM Modulation of the miR-203/Bcl-w apoptotic axis [80]
MGC 803 0–60 µM Modulation of MAPK-signaling pathways [82]
Molecules 2021, 26, 7368 13 of 19

Table 2. Cont.

Cancer Type Experimental Model (s) Dose Proposed Mechanism (s) References
HepG2 0, 50 and 100 µM Inhibition of cyclin D1 expression [83]
Suppression of glutamine uptake, Inhibition
Hep3B, BEL-7404 50–125 µM [84]
of SLC1A5
Liver cancer
Induction of G1 phase cell cycle arrest in
HepG2, Huh-7 30–120 µM [85]
cancer cells
Modulation of the expression of multiple
SNU-182, Hep3B, HepG2 10–100 µM [86]
tumorigenesis-related gene proteins
Induction of apoptosis,
KB 0, 0.1 and 1 µg/mL [91]
Enhancement of caspase-3 and -7 activities
Oral cancer
C666-1, HONE1, & HK1 0–50 µM Inhibition of STAT3 activation [92]
HONE1 0–300 µM Inhibition of STAT3 activation [93]
Inhibition of the caspase-1/IL-1 inflammatory
Saos-2, MG-63 0–100 µM [95]
Bone Cancer signaling axis
MG-63 0–80 µM Induction of apoptosis in cancer cells [96]
Glioblastoma
U251, U87 100 µM Induction of autophagy [103]
cancer
Inhibition of inflammatory cytokine
U251, U87 50 µM, 100 µM [107]
caspase-1 activation
Inhibition of MMP1, MMP13, uPA, and
Skin cancer A375.S2 0–2 µM [110]
Ras expressions
Inhibition of cancer cell proliferation,
A431 0–100 µg/mL [111]
Induction of apoptosis
Down-regulation of p-PI3K, p-AKT expressions,
B16 5–160 µM [112]
Up-regulation of RARβ and RARγ expressions
LNCaP, DU-145 20–400 µM Inhibition of VEGF and HIF-1α expressions [118]
LNCaP, 22Rv1, Decrease of cellular testosterone synthesis in a
Prostate cancer 12.5–50 µM/L [119]
PC3M, PC3 dose-dependent manner
LNCaP, 22Rv1, PC3 0–100 µM Suppression of androgen receptor signaling [120]

4.11. Prostate Cancer


Hypoxia and ionizing radiations (IR) were used to treat the prostate cancer cell
lines LNCaP and DU-145 with or without BBR therapy [118]. LNCaP cells were also
xenografted into naked mice and treated with IR or BBR. BBR improved the radiation
sensitivity of prostate cancer cells and xenografts in a dose-dependent manner, which
was linked to inhibition of the expression of HIF-1 and VEGF [118]. BBR suppressed
proliferation of the human prostate carcinoma epithelial cell line 22Rv1 and decreased
cellular testosterone synthesis in a dose-dependent manner [119]. BBR inhibited the activity
of the C3 enzyme from the Aldo-keto reductase family 1, rather than affecting mRNA or
protein expression [107]. BBR will thus join the active core of aldo-keto reductase family
1 member C3 and form an association with the amino acid residues Phe306 and Phe311,
according to molecular docking studies. Finally, the association of BBR with the aldo-
keto reductase family 1 member C3 inhibits 22Rv1 prostate cancer cell development by
inhibiting this enzyme and by reducing intracellular androgen synthesis [119].
In addition, BBR inhibited the androgen receptor (AR) transcriptional function in
castration-resistant prostate cancers (CRPC). BBR has little effect on the expression of AR
mRNA but causes AR protein degradation. Several ligand-binding domains truncated
AR splice variants have been discovered, and these variants are thought to help patients
develop CRPC. Surprisingly, these variants were found to be more vulnerable to BBR-
induced degradation than full-length AR. BBR also impairs the development of LNCaP
xenografts in nude mice and decreases AR expression in tumors, while normal prostate
morphology and AR expression are unaffected [120]. BBR has been shown to suppress
the capacity of prostate cancer cells to spread and infiltrate, these cells being strongly
Molecules 2021, 26, 7368 14 of 19

metastatic [49]. The inhibitory activity of BBR resulted in a substantial reduction in the
expression of a panel of mesenchymal genes that control developmental EMT. High BMP7,
NODAL, and Snail gene expression in metastatic prostate cancer tissues is associated with
shorter survival in patients with prostate cancers and offers potential therapeutic targets
among the EMT-related genes downregulated by BBR [49] (Table 2).

4.12. Thyroid Cancer


BBR inhibited RET expression in medullary thyroid carcinoma (MTC) cells by more
than 90% at a concentration of 2.5 µg/mL but did not affect TPC1 cells [121]. Canadine,
a structural analog of BBR, did not affect RET expression in MTC TT cells and had little
interaction with the RET G-quadruplex. In TT cells, the BBR-mediated downregulation
of RET inhibits cell proliferation by causing cell cycle arrest and activation of apoptosis,
as evidenced by a two-fold increase in caspase-3 activity and downregulation of cell
cycle regulation [122]. Two thyroid cancer cell lines, 8505C and TPC1, exhibited a dose-
dependent growth decrease upon BBR treatment. Following BBR treatment, 8505C cells
displayed a significant increase in apoptosis, whereas TPC1 cells showed cell cycle arrest
at the G0/G1 phase [123]. After BBR therapy, immunoblots of p-27 expression revealed
BBR caused a mild upregulation of p-27 in 8505c cells but a moderate upregulation of p-27
in TPC1 cells [123].

5. Conclusions
Cancer is a large category of disease that severely affects people’s health. Thus, there is
a vital need for cancer prevention and treatment advancement. Surgery, radiotherapy, and
chemotherapy are the most often used approaches to cancer care. People may also abandon
anticancer treatments due to their ineffectiveness and adverse side effects, resulting in
the illness progression and reduced overall survival rate. Resistance to anticancer drugs
may be conferred by target alteration, drug-efflux pumps, increased cellular tolerance to
apoptosis, increased DNA harm tolerance to therapy, reparability, and enhanced neoplastic
proliferation. Resistance may be due to improvements in the stroma and tumor climate
as well as cancer microenvironments. Cancer cells utilize a number of these pathways,
complicating clinical strategies for each patient. Recent advancements in cancer care, such
as selective and immunotherapy, also provided substantial benefits.
However, during the last decade, several clinical studies and lab analyses have been
conducted to investigate the efficacy of BBR in curing cancer. Additionally, BBR was found
to control pro and anticancer miRNAs and lncRNA levels and has been shown to improve
the effectiveness of chemotherapy and radiation therapy. However, BBR’s direct cytotoxic
impact is not considered very powerful. It acts at concentrations sometimes greater than
100 µM for certain cancer cell lines. Nevertheless, the cytotoxic action of BBR is moderate
as it ranges from 10 to 100 µM. BBR’s slow absorption, efflux from intestinal cells by
P-gc, and comprehensive metabolism by intestinal and hepatic cells render difficult its use
in vivo. Consequently, progresses must be made on developing both the pharmacokinetic
profile and the anticancer efficacy of BBR in the future. As BBR shows promising efficacy
concerning anticancer potential, it may be a potential candidate in innovative anticancer
drug discovery.

Author Contributions: Conceptualization, A.R. and M.I.; methodology, A.R., M.I., Z.A.S., T.A.-I. and
T.B.E.; software, A.R. and M.I.; validation, A.R., M.I., Z.A.S., T.A.-I. and T.B.E.; formal analysis, S.M.,
Z.K., F.A.A., A.S.M.A., I.K., M.M.R., P.J. and T.A.G.; investigation, A.R. and M.I.; resources, S.M., Z.K.,
F.A.A., A.S.M.A., I.K., M.M.R., P.J. and T.A.G.; data curation, A.R., M.I., Z.A.S., T.A.-I. and T.B.E.;
writing—original draft preparation, A.R., M.I., T.B.E., A.A.K. and S.M.; writing—review and editing,
S.M., Z.K., F.A.A., A.S.M.A., I.K., M.M.R., P.J. and T.A.G.; visualization, A.R. and M.I.; supervision,
A.R. and P.J.; project administration, A.R. and P.J.; funding acquisition, A.R. and P.J. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Molecules 2021, 26, 7368 15 of 19

Institutional Review Board Statement: Not applicable.


Informed Consent Statement: Not applicable.
Data Availability Statement: Available data are presented in the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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