Synthesis of a novel fluorescent berberine derivative convenient for its subcellular localization study

Article abstract of DOI:10.1016/j.bioorg.2020.104021

Berberine is a naturally occurred isoquinoline alkaloid that shows great potential for developing anticancer drugs. However, the problem stays of poor understanding of the mechanisms of anticancer action of berberine. It depends on evaluation of berberine

Full text of DOI:10.1016/j.bioorg.2020.104021

Journal Pre-proofs
Synthesis of a novel fluorescent berberine derivative convenient for its subcel-
lular localization study
Meng Jin, Xiuna Ji, Rostyslav Stoika, Kechun Liu, Lizhen Wang, Yang Song
PII:
S0045-2068(20)31318-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.104021
YBIOO 104021
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
21 January 2020
21 April 2020
13 June 2020
Please cite this article as: M. Jin, X. Ji, R. Stoika, K. Liu, L. Wang, Y. Song, Synthesis of a novel fluorescent
berberine derivative convenient for its subcellular localization study, Bioorganic Chemistry (2020), doi: https://
doi.org/10.1016/j.bioorg.2020.104021
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Synthesis of a novel fluorescent berberine derivative convenient for its
subcellular localization study
Meng Jin , Xiuna Ji , Rostyslav Stoika , Kechun Liu , Lizhen Wang a,b,*, and Yang Song d
a,b
a,b
c
a,b
aBiology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353,
Shandong Province, China
bEngineering Research Center of Zebrafish Models for Human Diseases and Drug Screening of
Shandong Province, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353,
Shandong Province, China
cDepartment of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, National
Academy of Sciences of Ukraine, Lviv, Ukraine
dBiological Engineering College, Qilu University of Technology (Shandong Academy of Sciences),
Jinan 250056, Shandong Province, China
*Corresponding author. Tel.: +86 0531 82605331; fax: +86 0531 82605331
E-mail: wlzh1106@126.com
Abstract
Berberine is a naturally occurred isoquinoline alkaloid that shows great potential for developing
anticancer drugs. However, the problem stays of poor understanding of the mechanisms of anticancer
action of berberine. It depends on evaluation of berberine’s pharmacokinetics, namely monitoring of
its uptake and distribution in cells, tissues and organs. In order to address these problems, we have
designed and synthesized a novel berberine derivative BBR-BODIPY bearing a fluorescent tag that
allows screening its interaction with the targeted cells. It was shown that the synthesized fluorescent
derivative could penetrate into human breast carcinoma MCF7 cells, and then induced apoptosis
detected by the Western Blot analysis as changed expression of apoptosis-related proteins, including
Bax, Bcl2, and Cyto C released from mitochondria, Cleaved Caspase 9, Cleaved PARP, Pro-Caspase
3
, and Cleaved Caspase 3. The results of MitoTracker analysis followed by the confocal microscopy
of sub-cellular localization of BBR-BODIPY in the MCF7 cells demonstrated excellent
cell-penetrating ability of this compound even at low concentrations, and mitochondria was the main
site of its accumulation. Together with the results of Western Blot analysis, these data indicated that
the mitochondria pathway might be involved in berberine-induced apoptosis.
Keywords: Fluorescent probe; fluorescently labeled natural product; berberine; anticancer
1

1
. Introduction
Berberine, a naturally occurred isoquinoline alkaloid isolated from Rhizoma coptidis,1-3 has been
drawn great attention due to its various biological activities, including anticancer,4-6 antimalarial,7
anti-Alzheimer's disease,8-11 antiviral, antidiabetic,
12
13-15
antileishmanial, and cholesterol lowering
16
effect.17 During the past several years, many research groups have focused on the anticancer
investigation of berberine and its analogs. It has been proved that berberine can induce apoptosis and
autophagy of many cancer cell lines, inhibit proliferation and migration of cancer cells, and arrest
their cell cycling.4,18-24 However, the molecular mechanisms of its anticancer activity remain largely
unknown. Some researchers proved that berberine could bind with telomeric G-quadruplex, a special
structure unit presented in the telomeric DNA, thereby leading to the apoptosis of cancer cells.25,26
While other researchers indicated that berberine significantly affected the mitochondrial membrane
potential and induced the expression of cell apoptosis proteins, which further resulted in cell
apoptosis.27-29 Therefore, it is important to investigate the cellular targets of berberine and underlying
molecular mechanisms that might be critical for developing novel berberine-based anticancer drugs.
Recently, several methods have been developed for investigating cellular targets of drugs or
biomolecules,30 such as two-dimensional gel electrophoresis, gene chip technology, and protein
microarrays. However, these methods usually need complex manipulation procedure for indirect
analyses. In contrast, fluorescently labeled probes demonstrate great potential in this area, as we can
directly visualize the cellular uptake and biological processes of drugs in living systems using a
fluorescence microscope.31-37 Using this technology, many fluorescently labeled natural products
have been synthesized and successfully applied for visualizing the complex drug-uptake/target
through live-cell/tissue imaging or in-gel fluorescence scanning.32-36,38,39
This paper reported the synthesis and biological evaluation of a novel fluorescently labeled
berberine derivative BBR-BODIPY. The cellular uptake and mitochondria targeting ability of this
derivative were investigated in MCF7 cell line through a fluorescence microscope, and the
expression of various proteins related to cell apoptosis was studied by Western-blot analysis. This
method is very convenient for inspecting berberine’s traffic in the targeted cancer cells, which is
important for better understanding the molecular mechanism of its anticancer activity and very
helpful for developing novel berberine-based anticancer drugs.
2. Experimental
2
.1 General procedures
Berberine and sodium ascorbate were obtained from TCI Development Co., Ltd. without any
2

treatment. 1,6-Dibromohexane, boron(tri)fluoride etherate, 2,3-dicyano-5,6-dichlorobenzoquinone
and 2,4-dimethylpyrrole were bought from Shanghai Macklin Biochemical Co., Ltd.. Other solvents
and reagents were all commercially available and used without any further purification.
1
13
Determination of H NMR and C NMR spectra was finished using a Bruker AV 400 spectrometer.
Measurement of the high-resolution mass spectra (HRMS) was accomplished by an Agilent QTOF
6
520 mass spectrometer. Ultraviolet-visible (UV-vis) absorption spectrum was recorded on a
Shimadzu UV-vis spectrophotometer UV-2600. Fluorescence spectra was measured using a Hitachi
F-4600 spectrophotometer with a scan rate of 2400 nm/min. Cell Counting Kit-8 (CCK8) was
purchased from Dojindo.
2
.2 Synthesis of derivative BBR-BODIPY
Synthesis of compound 236
The solution of 4-hydroxybenzaldehyde (2.0 g, 16.39 mmol), 1,6-dibromohexane (12.0 g, 49.17
mmol) and K CO (6.8 g, 49.17 mmol) in 200 mL of acetonitrile was stirred under reflux for 2 days.
2
3
After TLC (petroleum ether/ethyl acetate, 2/1, v/v) showed the complete conversion of the starting
material, the mixture was diluted with 200 mL of ethyl acetate and filtered. The filtrate was washed
with water (150 mL × 3), and the organic layer was dried with absolute sodium sulphate. The solvent
was evaporated and the resultant residue was purified by column chromatography using petroleum
ether/ethyl acetate as eluent to give compound 1 as colorless oil. Compound 1 described above was
then dissolved in 150 mL of DMF and stirred at room temperature. To the mixture was slowly added
a solution of NaN (3.2 g, 49.17 mmol) in 10 mL of water, and the reaction was stirred at room
3
temperature for 6 h. After TLC (petroleum ether/ethyl acetate, 1/1, v/v) showed the disappearance of
the starting material, the reaction was poured into 300 mL of water and extracted with ethyl acetate
(
100 mL × 3). The organic phase was dried and evaporated, and the resultant residue was subjected
to column chromatography using ethyl acetate as eluent to give compound 2 as yellow oil (2.71 g,
7%, two steps).
6
Synthesis of compound 4
To a deoxygenated solution of compound 2 (400 mg, 1.62 mmol) and 2,4-dimethylpyrrole 3
(
308 mg, 3.24 mmol) in 300 mL of CH Cl was added TFA (154 mg, 1.35 mmol) at room
2 2
temperature. The reaction was stirred overnight to give a red solution. Then, to the solution was
added DDQ (368 mg, 1.62 mmol), and the reaction was stirred at room temperature for 30 min. After
which, 6 mL of Et N and 3.6 mL of BF •Et O were added, and the mixture was stirred for another 40
3
3
2
min. The organic layer was separated, dried with anhydrous magnesium sulfate, and evaporated. The
3

resultant residue was purified with column chromatography using CHCl as eluent to provide probe 4
3
1
as an orange-red solid (324 mg, 43%, three steps). H NMR (400 MHz, CDCl ): δ 7.17 (s, 1 H, ArH),
3
7
.14 (s, 1 H, ArH), 7.00 (s, 1 H, ArH), 6.98 (s, 1 H, ArH), 5.97 (s, 2 H, pyrrole-H), 4.01 (t, J = 6.4
Hz, 2 H), 3.31 (t, J = 6.8 Hz, 2 H), 2.55 (s, 6 H), 1.88‒1.81 (m, 2 H), 1.70‒1.63 (m, 2 H), 1.57‒1.47
1
3
(
m, 4 H), 1.43 (s, 6 H); C NMR (100 MHz, CDCl ): δ 159.63, 155.23, 143.16, 129.19, 126.95,
3
1
21.08, 115.06, 67.88, 51.40, 46.81, 29.14, 28.82, 26.58, 25.72, 14.58, 8.65; ESI-TOF HRMS (m/z):
+
+
calcd for C H BF N O , [M + H] , 466.2584; found, 466.2588.
2
5
31
2
5
Synthesis of compound BBR-BODIPY
Berberine derivative 513 (100 mg, 0.25 mmol) and compound 4 (116 mg, 0.25 mmol) were
dissolved in the mix solution of methanol (10 mL) and DMF (10 mL), to the mixture was added
CuSO ·5H O (33 mg, 0.13 mmol) and sodium ascorbate (26 mg, 0.13 mmol). The reaction was
4
2
o
heated to 50 C and stirred at ambient temperature for 6 hours. After TLC showed the conversion of
the starting materials (dichloromethane/methanol, 5/1, v/v), the solvent was evaporated and the
resultant residue was subjected to column chromatography to give BBR-BODIPY as an orange-red
1
solid (133 mg, 62%). H NMR (400 MHz, DMSO-d ): δ 9.70 (s, 1 H, CH=N), 8.93 (s, 1 H, ArH),
6
8
7
.31 (s, 1 H, ArH), 8.23 (d, J = 9.2 Hz, 1 H, ArH), 8.01 (d, J = 8.8 Hz, 1 H, ArH), 7.79 (s, 1 H, ArH),
.23 (d, J = 8.4 Hz, 2 H, ArH), 7.09 (s, 2 H, ArH), 7.06 (s, 1 H, ArH), 6.16 (s, 3 H), 5.46 (s, 2 H, –
OCH O–), 4.91 (t, J = 5.2 Hz, 2 H, CH ), 4.35 (t, J = 6.8 Hz, 2 H), 4.10 (s, 3 H, OCH ), 3.97 (t, J =
2
2
3
6
.0 Hz, 2 H, CH ), 3.63 (s, 1 H), 3.21‒3.16 (m, 2 H), 2.44 (s, 4 H), 1.81‒1.74 (m, 2 H), 1.69‒1.62
2
1
3
(
m, 2 H), 1.38 (s, 6 H), 1.20‒1.14 (m, 2 H); C NMR (100 MHz, DMSO-d ): δ 159.70, 155.11,
6
1
1
5
51.36, 150.36, 145.70, 143.16, 142.05, 137.92, 133.38, 131.09, 129.58, 127.08, 124.37, 122.41,
21.72, 120.83, 120.71, 115.59, 108.93, 105.88, 102.58, 73.49, 72.68, 72.43, 67.96, 67.02, 57.54,
5.87, 49.78, 30.05, 28.85, 26.86, 25.96, 25.38, 14.60; ESI-TOF HRMS (m/z): calcd for
+
+
C H BF N O , [M – Cl] , 825.3742; found, 825.3746.
4
7
48
2
6
5
2
.3 Biological investigations
Cell lines and culture conditions
MCF7 and MDA-MB-231 cell lines were obtained from ATCC (Manassas, VA, USA) and were
cultured in DMEM high glucose medium (Hyclone, Logan, UT, USA) containing 10 % fetal bovine
serum (FBS) and 1% penicillin/streptomycin (Hyclone, Logan, UT, USA) and incubated at 37 °C in
humidified air containing 5 % CO2.
4

CCK8 assay
Cell proliferation was evaluated by CCK-8 according to the protocol reported previously.40
4
Briefly, cells were seeded into 96-well plates in triplicate at a density of 110 cells per well. After
2 hours, 10 μL CCK8 medium were added into wells. After incubation for 3 h, the absorbance was
1
measured at a wavelength of 450 nm. The CCK8 assays were repeated three times.
Time-course and concentration-dependent imaging studies
Fluorescent microscopy was performed to observe the cellular uptake and localization of
BBR-BODIPY in a time- and concentration-dependent manner. Briefly, cells were seeded into
4
9
6-well plates at a density of 110 cells per well. The concentration-based cellular uptake study of
BBR-BODIPY was carried out by incubating in MCF7 cells with 2 μM, 4 μM, 8 μM, 16 μM of
BBR-BODIPY for 30 minutes. The time-based cellular uptake study of BBR-BODIPY was
performed by incubating in MCF7 cells with 8 μM of BBR-BODIPY for 5, 15, 30, and 60 min. The
cells were washed with PBS for three times and subjected to fluorescent microscopy (Zeiss, Jena,
Germany).
Subcellular Localization of BBR-BODIPY
4
In brief, 210 cells MCF7 cells were seeded in laser confocal Petri dish. After 24 h incubation,
cells were treated with BBR-BODIPY and a commercial available mitochondria targetable dye
MitoTracker and then incubated for 30 min at 37 °C. The cells were washed with PBS for three times
and subjected to laser confocal microscopy (Olympus, Tokyo, Japan).
Western blot analysis
Cells were harvested and collected in cold RIPA lysis buffer (Thermo Fisher Scientific,
Waltham, MA, USA) with protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MS, USA). The
supernatant was transferred to a new tube after centrifuge at 20,000 g for 10 min at 4 °C. Protein
concentration was determined by bicinchoninic acid protein assay (Thermo Fisher Scientific Pierce,
Waltham, MA, USA). Samples were boiled in 5×SDS-PAGE loading buffer (Beyotime, Shanghai,
China) and ready for western blotting. Western blotting was carried out according to the Bio-Rad
electrophoresis protocol. Primary antibodies used were -Actin (1: 40000, Sigma-Aldrich, St. Louis,
MS, USA), rabbit anti-Bax antibody (1:500, Cell Signaling Technology, Danvers, MA, USA), mouse
anti-Cyto C antibody (1:500, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Cleaved
Caspase 9 (1:500, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Procaspase 3 (1:200 ,
5

Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Cleaved Caspase 3 (1:200, Cell
Signaling Technology, Danvers, MA, USA), rabbit anti-Cleaved PARP (1:1000, Cell Signaling
Technology, Danvers, MA, USA), rabbit anti-Bcl2 (1:1000, Cell Signaling Technology, Danvers,
MA, USA). Secondary antibodies are HRP-conjugated donkey anti-rabbit IgG (for Bax, Cleaved
Caspase 9, Pro-caspase 3, Cleaved Caspase 3, Bcl2 and Cleaved PARP; 1:10,000, ZSGB-BIO,
Beijing, China) and HRP-conjugated donkey anti-mouse IgG (for β-Actin and Cyto C; 1:10,000,
ZSGB-BIO, Beijing, China) were respectively used. To estimate the molecular weight, a pre-stained
protein ladder (26616, Thermo Scientific, Waltham, MA, USA) was used. Western blots were
carried out in triplicate by using three different batch of harvested proteins, then normalized to

-Actin. The results were analyzed by one-way ANOVA followed by Bonferroni's post-hoc test in
GraphPad Prism software 7.0 and expressed as mean ± SEM. P<0.05 was considered as significant.
3
. Results and discussion
3
.1 Design and synthesis of fluorescently labeled berberine derivative
Despite that several strategies for constructing fluorescently labeled natural products are
available, the common concern is the size of the fluorescent tags. It should be small enough and have
no or little effect on the bioactivity of the synthesized products.41,42 In this work, the BODIPY dye
was used as the fluorescent tag due to its nontoxicity, as well as excellent photo- and
4
3
chemostability. Attaching of the fluorescent tag to 9-O-position of berberine was accomplished by
using click chemistry, which would form a triazole linker with high stability. In addition, previous
work had revealed that modification of berberine on its 9-O-position could be easily achieved and
had some positive effect on its anticancer activity.23,44,45
As depicted in Scheme 1, fluorescent tag 4 was synthesized from 4-hydroxybenzaldehyde,
which was reacted with 1,6-dibromohexane in the presence of K CO under mild conditions to
2
3
provide compound 1 with 79% isolated yield. Subsequent substitution of the bromine atom in 1 with
an azido group using NaN in DMF smoothly furnished compound 2 with 85% yield. Conversion of
3
2
into fluorescent tag 4 was finished in 3 steps with 43% total yields, including treatment of 2 with
trifluoroacetic acid (TFA), addition of DDQ, and then reaction with triethylamine (TEA) and boron
trifluoride diethyl etherate (BF •OEt ). Attaching of 4 with alkylated berberine derivative 5 was
3
2
accomplished using click chemistry in the presence of CuSO •5H O and sodium ascorbate, which
4
2
afforded the fluorescently labeled berberine derivative BBR-BODIPY with 62% yield.
6

Scheme 1. Synthesis of fluorescently labeled berberine derivative BBR-BODIPY
Br
N3
1
. 3, TFA, DCM
2
. DDQ
1
,6-dibromohexane
K2CO3
acetonitrile
OH
NaN3
DMF
3. Et3N and BF3 OEt2
43%, 3 steps
O
O
85%
79%
CHO
4-hydroxybenzaldehyde
N
H
CHO
CHO
1
2
3
O
Cl
N
N
N
N3
O
O
N
O
CuSO4 5H2O
sodium ascorbate
CH3OH
OCH3
62%
O
O
O
Cl
N
O
N
N
F
B
N
N
F
OCH3
B
F
F
5
4
BBR-BODIPY
3
.2 Anti-proliferative activity of berberine and its derivative
In order to estimate if the designed fluorescently labeled berberine has preserved its anticancer
activity, the anti-proliferative activity of BBR-BODIPY was investigated on different human breast
4
0
carcinoma cell lines including MCF7 and MDA-MB-231 by using CCK8 assay , as depicted in
Table 1. We first determined the IC of BBR-BODIPY and it is 40.81 ± 1.61 μM in MCF7 cells and
5
0
4
1.46 ± 1.62 μM in MDA-MB-231 cells. While the IC50 of berberine itself in MCF7 cells and
MDA-MB-231 cells were 98.07 ± 1.99 μM and 122.00 ± 2.09 μM, respectively. The higher
anti-proliferative activity of the BBR-BODIPY than that of the berberine indicated that
BBR-BODIPY could be used for further studies.
Table 1. Anti-proliferative activities and fluorescent property of berberine, BBR-BODIPY and probe 4
a
0.5% DMSOb
λex (nm) λem (nm)
Cell lines (IC , μM)
5
0
Compounds
MCF7
98.07 ± 1.99
40.81 ± 1.61
> 100
MDA-MB-231
122.00 ± 2.09
41.46 ± 1.62
> 100
Berberine
BBR-BODIPY
4
―
―
490
480
525
505
aCCK8 assays: Cells were treated with different concentrations (1 μM, 2 μM, 4 μM, 8 μM, 16 μM and 32 μM) of
berberine and BBR-BODIPY for 24 h. Data are presented as mean ± SEM (in triplicate).
bFluorescent property was evaluated in 0.5% DMSO aqueous solution with a concentration of 1 × 10–5 M, λex:
maximum excitation, λ : maximum emission.
em
7

3
.3 Fluorescent Property
The fluorescent property of BBR-BODIPY was evaluated in 0.5% DMSO aqueous solution
with a concentration of 1 × 10–5 M. The BBR-BODIPY probe showed maximum excitation
wavelength at 490 nm and maximum emission wavelength at 525 nm (Figure 1 and Table 1), which
indicated its large stokes shift for bio-imaging studies.
Figure 1. Normalized absorption (black) and emission (red) spectra of 1 × 10–5 M solution of
BBR-BODIPY in 0.5% DMSO aqueous solution.
3
.4 Cellular uptake investigation
The concentration-dependent uptake of BBR-BODIPY was studied by incubating MCF7 cells
for 30 min in culture medium with different concentrations (2 μM, 4 μM, 8 μM and 16 μM) of
BBR-BODIPY. Such concentrations had been selected because they were all below the IC value of
5
0
the BBR-BODIPY (Table 1). As shown in Figure 2A, the fluorescence intensity was significantly
enhanced with the concentration increasing from 2 μM to 8 μM. When the concentration reached up
to 16 μM, no obvious fluorescence changes could be observed. These results indicate that probe
BBR-BODIPY exhibited excellent cell-penetrating ability and could be taken up into MCF7 cells
even at very low concentration (2 μM). Study of time-dependent uptake of BBR-BODIPY was also
performed in MCF7 cells and the results were shown in Figure 2B. Clearly, a green fluorescence
could be observed in MCF7 cells when they were incubated with 8 μM of BBR-BODIPY for 5 min,
and the fluorescence intensity demonstrated distinguishable improvement with the time passing.
After 30 min incubation, the fluorescence intensity was enhanced very slowly. Thus, our findings
revealed that fluorescently labeled berberine derivative could be quickly taken up into MCF7 cells
already in 5 min, and the fluorescence intensity exhibited a time-dependent manner.
8

Figure 2. Cellular uptake of BBR-BODIPY probe by MCF7 cells. (A) Concentration-dependent
uptake study of BBR-BODIPY in MCF7 cells: 2 μM (a), 4 μM (b), 8 μM (c), 16 μM (d) of
BBR-BODIPY applied for 30 min; (B) Time-dependent uptake study of BBR-BODIPY in MCF7
cells: 8 μM of BBR-BODIPY applied for 5 min (a), 15 min (b), 30 min (c) and 60 min (d). Scale bar
is 50 μm.
To verify that the cellular uptake of BBR-BODIPY probe was driven by berberine instead of
probe, MCF7 cells were incubated with 8 μM of probe 4 for 3 h. As shown in Figure 3A, very weak
fluorescence could be observed after 3 h of such incubation, which indicated that probe 4 exhibited
poor cell-penetrating ability, because good cell-penetrating ability of probe 4 should induce a strong
green fluorescence in cells. More importantly, this information also proved that the penetration of
BBR-BODIPY into MCF7 cells was mainly driven by berberine but not probe 4. In addition, MCF7
cells were also incubated for 3 h with 8 μM of berberine, in order to exclude a possibility that the
fluorescence was produced by berberine itself. As a result, berberine could not induce any
fluorescence enhancement after 3 h incubation (Figure 3B).
9

Figure 3. Microscopic images of cellular uptake of probe 4 (A) and berberine (B) in MCF7 cells
treated for 3 h. Scale bar is 50 μm.
3
.5 Sub-cellular localization of fluorescently labeled berberine derivative
The sub-cellular localization of fluorescently labeled berberine derivative BBR-BODIPY in
MCF7 cells was investigated by co-staining cells with BBR-BODIPY and commercially available
MitoTracker Red (a mitochondria targetable dye). To clearly visualize the fluorescent images, the
optimized concentration was selected as 8 μM, and the duration of incubation was 30 min. Then,
cells were imaged using a confocal laser scanning microscopy. The sub-cellular localization of
BBR-BODIPY was displayed as green fluorescence depicted in the left column of Figure 4. The
localization of mitochondria in cytoplasm was presented as strong red fluorescence produced by the
MitoTracker Red (right column in Figure 4). Clearly, after 30 min of incubation, the overlap of the
fluorescence of BBR-BODIPY and MitoTracker Red showed a yellow fluorescence (middle column
in Figure 4A) with a Pearson’s colocalization coefficient of 0.72 (Figure 4D, analyzed by ImageJ
software), thus implying the accumulation of berberine derivative BBR-BODIPY in mitochondria.
The yellow fluorescence was significantly enhanced after 6 h of incubation (Figure 4B) and the
Pearson’s colocalization coefficient was 0.88, which indicated the large accumulation of
BBR-BODIPY in mitochondria. The sub-cellular localization of probe 4 was also investigated as a
control group (Figure 4C). Obviously, its sub-cellular localization was displayed as weak green
fluorescence after 6 h of incubation, which proved again its poor MCF7 cell penetrating ability.
Furthermore, the green fluorescence of probe 4 and red fluorescence of MitoTracker Red could be
easily distinguished and exhibited almost no overlap, which indicated that probe 4 was mainly
localized in the cytoplasm of MCF7 cells. Based on our above results that uptake of BBR-BODIPY
into MCF7 cells was mainly driven by berberine, we could conclude that mitochondria might be the
main organelle for accumulation of berberine.
1
0

Figure 4. Microscopic images of cellular uptake of BBR-BODIPY and MitoTracker (a commercial
available mitochondria targetable dye) by MCF7 cells after incubation for 30 min (A) and 6 h (B);
Microscopic images of cellular uptake of probe 4 and MitoTracker by MCF7 cells after incubation
for 6 h (C); Average colocalization coefficients of BBR-BODIPY and MitoTracker in MCF7 cells
(D). Cells were imaged using a confocal laser scanning microscopy. Scale bar is 10 μm.
3
.6 Investigation of mechanism underlying potential anticancer activity of BBR-BODIPY
It has been reported that mitochondria plays a pivotal role in the intrinsic pathway of apoptosis.46
Taking in consideration the anti-proliferative activity and mitochondria accumulation of
BBR-BODIPY, we hypothesized that berberine induced the apoptosis of cancer cells mainly through
the mitochondria pathway. Therefore, the expression of various proteins that had been proved to play
a critical role in controlling the mitochondrial pathway was investigated through Western Blot
analysis. Bax and Bcl2 are well-known proteins regulating mitochondria pathway. Over-expression
of Bax or down-regulation of Bcl2 can change the mitochondrial membrane potential which results
in apoptosis of cancer cells;47,48 Caspase 3 is one of the apoptosis protein mediator related to
mitochondria pathway, and over-expression of Caspase 3 can change the cell morphology and lead to
1
1

4
9
apoptosis; PARP is among critical substrates of Caspase 3, and the expression of cleaved forms of
PARP is usually enhanced during apoptosis of cancer cells.50 As demonstrated in Figure 5, the
amounts of Bax and Cyto C released from mitochondria, and cleaved Caspase 9 were significantly
up-regulated in MCF7 cells treated with both berberine and BBR-BODIPY. The expression of Bcl2
and Pro-caspase 3 proteins were apparently decreased, which was accompanied with the increased
amount of cleaved Caspase 3 and its substrate, cleaved PARP. In contrast, MCF7 cells treated with
probe 4 could hardly show obvious changes in expression of mitochondrial apoptosis-related
proteins, which proved that the mitochondria-dependent pathway of apoptosis was mainly induced
by berberine (Figure 6). Notably, the variation in protein levels induced by BBR-BODIPY was
larger than that induced by free berberine, suggesting that modification of berberine with probe 4
could enhance its anticancer activity.
Figure 5. (A) Results of Western Blot analysis of affecting by BBR-BODIPY, probe 4 and
berberine (BBR) of the expression of mitochondrial apoptosis-related proteins, including Bax, Bcl2,
released Cyto C, cleaved Caspase 9, cleaved PARP, pro-Caspase 3, and cleaved Caspase 3. -Actin
was used as the loading control. MCF7 cells were treated with the studied compounds for 12 h. (B-H)
The density ratio of the amount of studied proteins to -Actin shown as relative expression units.
Data are presented as mean ± SEM. *P <0.05, **P <0.01, ***P <0.001 vs Control.
1
2

Figure 6. Scheme demonstrating a mechanism of induction of apoptosis in treated cells by
BBR-BODIPY through mitochondrial pathway. Puma–p53 upregulated modulator of apoptosis,
Cyto C–Cytochrom C released to cytoplasm from mitochondria, Apaf 1–Apoptotic protease
activating factor 1, Cas 9–Caspase 9, Cas 3–Caspase 3.
4
. Conclusions
This paper describes development of a novel fluorescently labeled berberine derivative and
demonstrates its potentials at being used for revealing mechanisms of the anticancer activity of
berberine. Investigation of cellular uptake revealed that low concentrations of probe BBR-BODIPY
could be rapidly (5 min) taken up into human breast carcinoma cells of MCF7 line. More
importantly, most of the probes were accumulated in mitochondria of these cells, indicating that
berberine might induce cell apoptosis through mitochondria pathway which was further confirmed
by the results of conducted Western Blot analysis of apoptosis-related proteins. Overall, our study
provides a novel strategy for detection of the sub-cellular localization of berberine, which is a critical
issue at investigating mechanism of its anticancer activity. This strategy can be applied for
developing various fluorescently labeled natural products and drugs used for their cellular targeting
study and molecular mechanisms investigation.
Conflict of Interest
The authors declare no competing financial interests.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 81802629),
Young Doctoral Cooperation Foundation of Qilu University of Technology (Shandong Academy of
Sciences) (No. 201713SH2024), Natural Science Foundation of Shandong Province (No.
1
3

ZR2018PB007), and International Science and Technology Cooperation Program of Shandong
Academy of Sciences (No. 2019GHZD10). This work was also supported by the High-end Talents
Program of Jinan (No. 2019GXRC044), Jinan High-end Foreign Experts Recruitment Program (No.
2
019T10), and Young Science Foundation of Shandong Academy of Sciences (No. 2020QN0018).
Supplementary data
Supplementary data associated with this article, can be found in the online version, at
http://dx.doi.org/
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Highlights
1
. A fluorescently labeled berberine derivative was synthesized and used for investigating the
subcellular localization of berberine.
2
3
. The mitochondria pathway might be involved in berberine-induced apoptosis.
. The research provides a novel strategy that can be applied for studying the molecular
mechanisms of natural products.
Table of contents entry
Synthesis of a novel fluorescent berberine derivative convenient for its subcellular localization
study
Meng Jin, Xiuna Ji, Rostyslav Stoika, Kechun Liu, Lizhen Wang, and Yang Song
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