Discovery of mitochondria-targeting berberine derivatives as the inhibitors of proliferation, invasion and migration against rat C6 and human U87 glioma cells

Article abstract of DOI:10.1039/c4md00264d

This research aims to synthesize lipophilic berberine derivatives and evaluate their antiglioma effects on C6 and U87 cells. The introduction of substituents with various carbon chain lengths on C-13- or C-9-O-position of the berberine scaffold led to the discovery of several potent inhibitors against glioblastoma cells. Derivatives substituted with the carbon chains of moderate length (twelve carbons) displayed improved lipophilicity and the strongest inhibitory effects. Several compounds presented dose-dependent repression against proliferation (IC₅₀, 1.12-6.12 μM) and blocked migration and invasion by over 60% at lower dose levels. Furthermore, preliminary research about the underlying mechanism for the enhanced antiglioma ability indicated that these analogues preferentially localized into mitochondria, inducing the up-regulation of ROS production. Overall, these compounds represent promising candidates to combat glioblastoma and highlight new insight into the antiglioma therapy through interaction with mitochondria. This journal is

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Discovery of mitochondria-targeting berberine derivatives as inhibitors
ofproliferation, invasion and migration against rat C6 and human U87
glioma cells
a,c,d
a,d
a
a
b
b
b
5
0
5
Shengnan Fu, Yanqi Xie, Jue Tuo, Yalong Wang, Wenbo Zhu, SihanWu, Guangmei Yan, and
Haiyan Hu*
,a
a
School of Pharmaceutical Sciences, Sun Yat-sen University, Waihuan East Road 132, Guangzhou
10006, China
5
b
1
Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Zhongshan II
Road 74, Guangzhou 510080, China
c
Department of Pharmacy, The People’s Hospital of ZhongShan, Sunwen East Road 2, Zhongshan
28403, China
5
*
Corresponding author, Tel: +86-020-39343118; Fax: +86-020-39343118
1
E-mail: lsshhy@mail.sysu.edu.cn
d
These authors contributed equally.
Abbreviations
GBM, Glioblastoma multiforme; MMP, matrix metalloprotease; Bclꢀ2, Bꢀcell CLL/lymphoma 2; TLC,thinꢀlayer chromatography; MTT,
(3,4,5ꢀdimethylthiazolꢀyl)ꢀ2,5ꢀdiphenyl tetrazolium; IC , half maximal inhibitory concentration.
2
0
50
Abstract
This research aims to synthesize lipophilic berberine derivatives and evaluate their antiglioma effects on
C6 and U87 cells. Introduction of substituents with various carbon chain lengths on Cꢀ13ꢀ or Cꢀ9ꢀOꢀ
position of the berberine scaffold led to the discovery of several potent inhibitors against glioblastoma
cells. Derivatives substituted with carbon chains of moderate length (twelve carbons) displayed improved
lipophilicity and the strongest inhibitory effects. Several compounds, presented doseꢀdependent
repression against proliferation (IC , 1.12ꢀ 6.12 µM) and blocked migration and invasion by over 60% at
2
5
5
0
lower dose levels. Further preliminary research about the underlying mechanism for the enhanced
antiglioma ability indicated that these analogues preferentially localized into mitochondria, inducing upꢀ
regulation of ROS production. Overall, these compounds represent promising candidates to combat
glioblastoma and highlight new sight into the antiglioma therapy through interaction with mitochondria.
3
0
Glioblastoma multiforme (GBM), a grade Ⅳastrocytoma, is the
[journal], [year], [vol], 00–00 | 1
Introduction
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1
16
most common and malignant primary brain tumor. Despite
literature. Absorbingly, berberine can selectively accumulate in
17
multiple therapies, GBM claims its sufferers’ lives typically less
than 14 months after diagnosis. One subtle characteristic of
tumor cell mitochondria, which could be attributed to its
structural amphiphilicity and delocalized positive charge.
2
18
GBM is the highly invasive behavior, conferring glioma cells the
Besides, mitochondrialꢀbased cancer cell apoptosis induced by
5
0
5
0
propensity of rapidly infiltrating neighbor normal tissue and 60 berberine has been confirmed in several nonꢀCNS malignant cell
3
making the complete surgical resection impossible. Actually,
after surgical removal of a glioma, 96% of the recurrent tumors
derived from the residual invasive glioma cells arise within 2 cm
lines, with the underlying mechanisms interpreted as decreasing
ATP levels and activating apoptotic cascades via lowering the
mitochondrial membrane potential, inhibiting complexⅠof the
4
from the resection cavity or immediately adjacent to the resection
respiration chain, induction of mitochondrial permeability
5
19
1
1
2
rim. Further, radiotherapy, also seems to have reached its 65 transition (MPT) and release proꢀapoptotic proteins to cytosol.
practical limit, due to at least partly inherent and/or acquired
radioresistance of highꢀgrade glioma. Moreover, traditional nonꢀ
selective chemotherapeutics such as temozolomide (an alkylating
agent) and irinotecan (a topoisomerase 1 inhibitor), have been
Further, recent investigations start to uncover berberine’s
potential in suppressing migration and invasion in nonꢀCNS
cancer cell lines. For example, berberine attenuates human gastric
tumor cell invasion and inhibits the metastatic potential of breast
6
reported that the survival advantage brought by these chemicals is 70 cancer cells through downꢀregulation of MMPs and Akt pathway
7
20
accompanied with certain observed severe side effects. In
modulation, respectively. However, current literature about
addition, some low molecular inhibitors of matrix
metalloproteases (MMPs) and antivascular pharmaceuticals were
investigated to suppress cancer invasion, yet only achieving
whether berberine derivatives could selectively interact with
mitochondria and repress the proliferation, migration and
invasion of malignant glioma cells is rare. In addition, a nonꢀ
75 ignorable obstacle that limits berberine’s clinical application is its
8
limited clinical results.
16
innate poor solubility and lipophilicity. A structureꢀactivity
relationship study on berberrubine derivatives indicated that
presence of lipophilic alkyl substituents of moderate length
Conclusively, the invasion and migration ability of glioma
cells remains to be the main reason of current unsatisfactory
therapies. Indeed, accumulated data substantiate that GBM
21
presents the optimal antibacterial activity, prompting us that the
invasion is regulated and influenced by extremely diversified and 80 lipophilicity alteration of berberine may favor its performance in
9
2
5
overlapping signal pathways instead of a single one. Hence, it is
extensively supported that future successful therapies in the
treatment of GBM will target multiple signal transduction
cascades, either by one drug aiming at the pivotal intersection of
several pathways or by a group of agents aiming at multiple
cytotoxicity and antiꢀmigration activity.
Previously, we have evaluated the antiglioma ability of four
berberine derivatives varying the alkyl chain length in the Cꢀ9ꢀOꢀ
position of berberine (3b-e) and found that they showed
85 inhibitory effects on C6 glioma cells in proliferation, migration
3
30
signaling pathways.
22
and invasion. In this study, five 13ꢀOꢀsubstitued berberine
analogs varying the alkyl chain length (5f-g) were synthesized
and characterized. However, to comprehensively compare the
antiglioma effects of ten berberine derivatives and achieve more
reliable results, in other words to avoid time interference on
experimental results, the MTT, migration and invasion assays
previously used to evaluated four 9ꢀOꢀsubstitued berberine
derivatives (3b-e) on C6 cells were performed again.
Simultaneously the same assays of another 9ꢀOꢀsubstitued
Coincidentally, dozens of lethal signaling pathways converge
on what seems to be a promising therapeutic target, the
1
0
mitochondrion organelle. Recent studies shed light on the crucial
role that mitochondria, at least partially, play in tumor invasion.
The high expression feature of BclꢀW (an mitochondrial outer
membrane antiꢀapoptotic protein) has been revealed in invasive
90
35
40
45
50
11
glioma cells in vivo and depletion of BclꢀW or BclꢀX mediated
L
by siRNA renders invasive glioma cells susceptible to cytotoxicꢀ
1
2
therapyꢀinduced apoptosis. Moreover, research showed that the 95 berberine derivative (3a) and five newly synthesized 13ꢀOꢀ
active Akt controlling the balance between cell survival and
substitued berberine derivatives (5f-g) on rat C6 glioma cells
were added. Additionally, human U87 glioma cells were chosen
to assess the total ten berberine derivatives in this study. The
underlying mechanisms for the improved antiglioma activity
13
apoptosis predominantly localized at the leading edge of
14
migrating cells, suggesting the implication between tumor
invasion and mitochondria. Hurd et al. have well validated the
dual effects of intracellular reactive oxygen species (ROS) 100 were preliminarily explored by studying the subcellular
mainly produced by mitochondriaon triggering signal pathways
localization of these compounds and measuring ROS production
of the C6 and U87 glioma cells with or without berberine
derivatives. The BBB penetration ability was also predicted since
these compounds have to function inside the brain.
1
5
for cell migration and invasion. Additionally, inhibition of heatꢀ
shock protein 90 (HSP90), which preferentially localizes in
malignant cell mitochondria but not in their counterparts of
10
normal cells, will reduce cell migration and invasion via
9
decreasing MMPꢀ9 expression in GBM. Therefore, it may be
1
05 Results and Discussion
possible to target mitochondria as the junction of multiple signal
cascades to achieve improved therapeutic efficacy for GBM
patients.
Chemistry
Synthesis and identification of 3a-e
Berberine (1), an isoquinoline alkaloid, possesses many
striking pharmacological effects elaborated in available
The synthetic pathway for compounds 3a-e and5f-j is illustrated
in Scheme 1. To obtain 3a-e, the first step involved pyrolysis of
55
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1
to the intermediate berberrubine(2). Following alkylation of the
respectively.
resulting 2 by corresponding alkyl bromides or the benzyl
bromide afforded five 9ꢀOꢀsubstitued berberine analogs. We
found the key determinant in synthesis of 9ꢀOꢀsubstitued
berberine derivatives was always keeping the reaction system
completely absent of oxygen. It should be noted that in the
alkylation step we simplified the reported condition of reflux at
Synthesis and identification of5f-j
5
15
To synthesize 5f-j, precursor1 was first converted into
intermediate dihydroberberine (4) bysodium borohydrideꢀ
promoted reduction. Subsequent sodium iodideꢀpromoted
enamine alkylation of 4 by alkyl bromides of various chain
lengths or the benzyl bromidefurnished five 13ꢀsubstitued
23
1
20 ℃ to stirring at 80 ℃ without compromising the
yield.Besides, the mass spectra of 3a-e show the molecular ions
at m/z 462, 490, 546, 574, and 412 (MꢀBr, ESIꢀMS), which are
1
0
1
40, 168, 224, 252 and 90 mass units higher than that of 2 (M+H),
2
0
Scheme 1. Synthesis of 3a-e, 5f-j. Reagents and conditions. (a) Decalin, N
2
, 190 ℃. (b) R
1 2 4
Br, DMF, N , stirring, 80 ℃. (c) NaBH , pyridine, stirring, rt. (d)
R
2
Br, anhydrous acetonitrile, NaI, N
2
, stirring, 80 ℃.
berberine analogues. This enamine alkylation step is
accompanied by restoring thearomaticity of one nitrogenꢀ
containing heterocycle, which is indispensible in maintaining the
planar characteristic of the berberine scaffold as well as the
pharmacological effects. The H NMR spectra indicate that five
compounds 5f-j share the same parent nucleus with 1.The singlet
reaction with pyridine as the solvent at room temperature for 20
min exceeded that of the reaction with the latter as the solvent at
0 ℃ for 8 h.
2
3
3
5
0
5
1
The first obstacle we met in synthesis of 5f-i was the
5
0
considerably low yields that could be ascribed to the following
2
5
reasons. Approximate 30%ꢀ40% of 4 converted back into 1 in
the enamine alkylation process which per se possesses the
drawbacks of low yields and numerous byproducts. The second
reason is that alkyl bromides with long carbon chains present
poor reaction activity. To improve the yield, we tried to optimize
the enamine alkylation in reaction solvent, pressure, temperature,
catalyst, feeding sequence and feeding ratio. Firstly, using ESIꢀ
MS monitoring the reaction process, we finally chose acetonitrile
to dissolve 4 instead of dichloromethane or DMF, which both
(
~8.95 ppm) displayed by the Cꢀ13 position proton of 1
1
disappears in the H NMR spectra of 5f-j, suggesting that the
substitution reaction occurs at Cꢀ13 position. Moreover, owing to
+
the electronꢀwithdrawing effect of Cꢀ7 position N , the
1
55
deshielding and higher field H chemical shifts of the protons in
1
Cꢀ13 substituents are also observed in the H NMR spectra of 5f-j,
similar with those of 3a-e.
Apart from the above approach to synthesize 5f-j, we
attempted another method including reduction of 1 to an 60 exhibited excellent solubility to 4 though, but the molecular ion
intermediate 8ꢀacetonyldihydroberberine and then alkylation of
this intermediate with bromides. Nonetheless, the yield of 8ꢀ
acetonyldihydroberberine was lower than that of the intermediate
peaks of target compounds failed to appear using the latter two as
solvents. Secondly, an elevated yield was achieved under high
pressure using an airtight thickꢀwalled flask as the reaction
2
4
4
0
23
4
, due to the former’s easy rearrangement to 1 during the heating
apparatus rather than the usual reflux method. Thirdly, we
process. In addition, during preparation of 4 we compared two 65 selected 80 ℃ as the reaction temperature since no significant
reaction solvents, pyridine and the methanol solution containing
potassium carbonate, in respect of reaction temperatures, time
and yields. Results manifested that the yield of 4 from the
improvement of the yield was attained at 100 ℃ and 130 ℃.
Fourthly, sodium iodide exhibited higher catalytic activity than
potassium iodide and the possible explanation could be the higher
4
5
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solubility of sodium iodide in acetonitrile. Finally, distinctive
feeding sequences and ratios were tried and the present sequence
was easily oxidized back to
Consequently, adding impure 4 to the following enamine
1
under aerobic conditions.
and ratio in the experimental section were the relatively 10 alkylation produced six byproducts per reaction, whose R values
f
advantageous combination in favor of the final yield.
were very close to those of target product in silica gelꢀbased TLC
Developing solvents: dichloromethane/methanol, 20:1, v/v).
Neither recrystallization nor silica gelꢀbased column or alumina
column chromatography could separate the total resulting
(
5
5
Another obstruction came down to purification of 5f-i. Highly
pure intermediate
4 was difficult to obtain from either
recrystallizationor chromatography since 4,as mentioned earlier,
26
1
mixture. After extensive attempts, we developed a threeꢀstep
approach to remove impurities. Firstly, most byproducts were
separated byneutral alumina column chromatography(Petroleum
ether/nꢀbutanol). Secondly, column chromatography on silica gel
IC50 (µM)
compounds Purity (%)
log P
C6
U87
1
98.89
99.12
99.08
96.23
99.16
96.80
98.17
96.75
95.62
95.49
99.46
ꢀ1.68 ± 0.24
2.26 ± 0.34
3.43 ± 0.21
3.41 ± 0.40
3.01 ± 0.40
ꢀ0.86 ± 0.16
2.37 ± 0.27
3.66 ± 0.30
3.24 ± 0.22
3.22 ± 0.31
ꢀ0.73 ± 0.14
> 20
> 20
(
nꢀbutanol/water/acetic acid) was harnessed to remove a
2
0
byproduct whose polarity was next to those of target products.
3
a
2.95 ± 0.22
1.24 ± 0.13
6.12 ± 0.64
5.73 ± 0.51
4.55 ± 0.28
15.2 ± 2.02
1
Finally, an unidentified impurity peak in H NMR spectra was
TM
successfully eliminated after the purification by Sephadex LHꢀ
3
b
2
0 column chromatography (Methanol).
3
c
3
d
4.45 ± 0.57 22.49 ±3.38
2
3
3
5
0
5
Log P values
3
e
11.48 ± 2.05
1.12 ± 0.35
1.65 ± 0.38
1.67 ± 0.46
2.37 ± 0.29
14.4 ± 1.48
> 20
In Table 1, the negative log P values of 1, 3e and 5j demonstrate
that introduction of benzyl to the berberine scaffold at the Cꢀ9ꢀ
Oposition and Cꢀ13 position will moderately augment the
lipophilicity of berberine, while alkyl substituents at both Cꢀ9ꢀO
and Cꢀ13 positions of berberine remarkably increase the
lipophilicity of berberine. Further, berberine derivatives with
moderate carbon chain length (3b and 5g) show the most
significant enhancement in lipophilicity, implying more
preferable transmembrane permeability than berberine.
5
f
3.65 ± 0.61
3.09 ± 0.54
3.25 ± 0.27
5.75 ± 0.68
> 20
5g
5h
5
i
5
j
a
Log P means the logarithm of the nꢀoctanol/water partition coefficients;
a
40 Purity of these compounds was determined by Highꢀperformance
LiquidChromatography. IC50: inhibitory concentration causing a 50%
reduction in cell proliferation in µM. Data were expressed as mean ± SD.
Table 1. Log P values, purity data , and effects on survival rate
b
(
IC , µM) of rat C6 and human U87 glioma cells by 1, 3a-e and
5
f-j
0
b
5
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Fugure 1. Survival rate (%) of C6 (A, C) and U87 (B, D) cells after incubated with different compounds at various concentrations for 24 h (n = 10). Data
are presented as mean ± standard deviation. P < 0.05; *versus control; **versus berberine.
5
25 instance, the survival rates are decreased to lower than 20% by
5
f-i at 10 µM (Figure 1C and 1D). However, 5j exerts the modest
Proliferation inhibition in MTT assays
repression effect on C6 and U87 cells at 20 µM since the survival
rates are as high as 37.24% and 67.20%, respectively. In general,
the MTT results of these synthesized derivatives on C6 cells are
in line with those of U87 cells and the C6 cells are more sensitive
to all the tested compounds than U87 cells are.
The antiproliferative effects of compounds 1, 3a-e and 5f-j was
assayed in rat C6 and human U87 glioblastoma cells using the
MTT ((3, 4, 5ꢀdimethylthiazolꢀyl)ꢀ2, 5ꢀdiphenyl tetrazolium)
assay that measures the number of metabolically active cells.
Figure 1 illustrates the relative survival rates of both cell lines
after incubation with various compounds at different dose levels
for 24 h. Firstly, exposure of C6 cells to 1 with dose up to 20µM
exhibits a minimal antiproliferative effect, while U87 cells are not
sensitive to 1 at this dose level (Figure 1A and 1B). The rest
derivatives 3a-e and 5f-j are active against both tumor cell lines
in a doseꢀdependent manner. Specifically, in Figure 1A and 1B,
3
0
1
1
2
0
5
0
Based on the MTT tests, IC50 values of the assayed compounds
were calculated and shown in Table 1. Of note, 3a-c and 5f-i are
superior to the clinically commonly used chemotherapeutics for
glioblastoma in relation to cytotoxicity. Indeed, the IC50 values of
temozolomide and irinotecan on U87 glioma cells are 19.38 µM
and higher than 250 µM, respectively. Recently,3b was
synthesized and also proved to be a more robust inhibitor on
human cancer HepG2 and HT29 cell lines. Moreover, data
trends in Figure 1 and Table 1demonstrate that the variation of
cytotoxic activity of the synthetic analogues correlates with
alternation of alkyl chain length. Namely, the presence of alkyl
chains with moderate length (12 carbons) at Cꢀ9ꢀOꢀposition or Cꢀ
35
27
3
a and 3b display the strongest antiꢀproliferation effects on both
28
cell lines, while 3d and 3e have a moderate effect. In particular,
3c achieves almost the same complete inhibition result against C6
cells as 3a and 3b do when the concentration of 3c reaches at 20
µM whereas the survival rate of U87 cells exposed to 3c is
40
3
8.95%. Further, for 13ꢀsubstituted berberine analogs, 5f-i also
1
3ꢀposition on the berberine scaffold guarantees the most
shows robust growth suppression on C6 and U87 tumor cells. For
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appreciable antiglioma activity. Nonetheless, introduction of the
benzyl group at aforementioned positions contributes to activity
elevation inconspicuously. These highly similar varying patterns
between the lipophilicity and cytotoxicity of compounds 1, 3a-e
and 5f-j led us to postulate that the elevated lipophilicity might
promote the transmembrane permeability of these compounds,
thus enhancing the intracellular concentrations and cytotoxicity.
Efficacy in transwell migration and invasion assays
1
0
As reported, berberine possesses potential repression on
migration and invasion in nonꢀCNS cancer cell lines. Hence, not
only to identify, but to compare the possible suppression of
berberine and its lipophiphilic derivatives on migration and
invasion of glioma cells, we tested 1, 3a-e and 5f-j using
transwell migration and invasion assays on Milipore cell culture
inserts with 8-µmpore size polycarbonate membranein C6 and
U87 cell lines. Migration and
5
15
Figure 2. Transwell migration (A) and invasion (B) assays in C6 cell lines (n=3). Dose levels for 1and3a-e are 5, 0.5, 0.5, 0.5, 0.5 and
0.5 ꢁM, respectively. Data are presented as mean ± standard deviation. P < 0.05; *versus control; **versus berberine.
20
invasion experiments were performed in the absence or presence
of matrigel on the polycarbonate membrane, respectively. To rule
out cytotoxicity as the driver of decreasing tumor cell migration
survival rates of all groups were higher than 85%. Second, the
number of migrated and invaded tumor cells to the lower
chambers was calibrated via being divided by the total cell
25
and invasion ability, first, we chose the minimum highest dose 30 numbers that were characterized through synchronous MTT
levels of all assayed compounds based on MTT assays so that all
assays at the same dose levels.
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Figure 3. Transwell migration (A) and invasion (B) assays in U87 cell lines (n=3). Dose levels for 1and3a-e are 5, 2.5, 2.0, 2.5, 2.5 and
.5 ꢁM, respectively. Data are presented as mean ± standard deviation. P < 0.05; *versus control; **versus berberine.
2
5
Figure 4. Transwell migration (A) and invasion (B) assays in C6 cell lines (n=3). Dose levels for 1and5f-j are 5, 0.5, 0.5, 0.5, 0.5 and 1
M, respectively. Data are presented as mean ± standard deviation. P < 0.05; *versus control; **versus berberine.
ꢁ
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As shown in Figure 2, 3, 4 and 5, all compounds exhibits
robust or moderate inhibitory effects against invasion and
5
Figure 5. Transwell migration (A) and invasion (B) assays in U87 cell lines (n=3). Dose levels for 1and5f-j are 5, 0.5, 0.5, 0.5, 2.5 and
.5 ꢁM, respectively. Data are presented as mean ± standard deviation. P < 0.05; *versus control; **versus berberine.
2
migration in both cancer cell lines except 13ꢀbenzylꢀsubtituted 35 cytotoxicity. Using these effective but low cytotoxic inhibitors to
berberine analog, 5j. A modest antiꢀmigration and antiꢀinvasion
activity is observed in the presence of berberine, which agrees
with previous proof that berberine could attenuate migration and
invasion in other tumor cell lines. Furthermore, 3d, 3e, 5i and 5j
display greater than 30% suppression and three other potent 40 taking.
derivatives (3b, 3c and 5g) block migration or invasion by over
60%. Overall, these experimental results substantiate that the
tested compounds are effective migration and invasion inhibitors.
block tumor cell metastasis will be more advantageous in longꢀ
term treatment of invasive tumor, since most chemotherapeutics
targeting cancer cell proliferation provoke acquired
chemoresistance and toxicity to healthy cells after longꢀtime
1
0
2
0
29
15
Subcellular localization in mitochondria
After comparing Figure 2A and Figure 3A, it could be
concluded that only those analogs with moderate alkyl chain
length (3band3c) could significantly lower tumor migration
tendency. Lengthening the carbon chain (3d) or shortening the
substitute (3a and 3e) will compromise the antiꢀmigration
potency. Likewise, the similar phenomenon is noted for 5f-j in
transwell migration assays (Figure 4A and Figure 5A).
Meanwhile, there is no cancer typeꢀspecific mode of action
revealed in migration inhibition evaluation. On the contrary, C6
and U87 cells are differently sensitive to the derivatives regarding
invasiveness. For example, the most powerful antiꢀinvasion
agents for C6 cells are 3c and 5f, whereas for U87 cells 3a, 3b
and 5g are the most remarkable ones (Figure 2B, Figure 3B,
Figure 4B and Figure 5B). Therefore, both substitutes with
suitable carbon chain length and tumor cell types influence the
activity of synthetic analogues in terms of subduing invasion
ability. Intriguingly, agents 1, 3d, 3e and 5j could attenuate
cancer cell migration and invasion without exert acute
To preliminarily examine the potential target of the synthetic
derivatives and if they preferentially localize into mitochondria as
berberine does, we selected four compounds (3b, 3c, 5g and 5h)
with high antigrowth, antiꢀmigration and antiꢀinvasion ability to
incubate with C6 and U87 cells at 1 µM dose level followed by
staining mitochondria with Mitotracker Green FM (500 nM) and
photographing. Berberine was also included as a control group.
Figure 6 depicts the laser confocal micrographs of doubleꢀstained
C6 cells (the left panel) and U87 cells (the right panel).
4
5
20
25
30
50
A perfect superposition between Mitotracker Green FM dye
images and those of berberine evidences the specific subcellular
localization of berberine to cell mitochondria, which is in
17
55
harmony with previous results. The overlapping photographs
for 3b, 3c, 5g and 5h unequivocally demonstrate that these
berberine derivatives have
a preference for mitochondrial
organelle.Such mitochondrionꢀtargeting feature could be
a
ascribed to two determinants. The primary reason is that the
8
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Figure 6. Laser confocal micrographs of doubleꢀstained C6 cells (Left panel) and U87 cells (Right panel). Notes: 1. Green channel: 500
nMMitotracker Geen FM stained mitochondria; 2. Yellow channel: 1 µM respective compounds; 3. Composite images of the former two
channels, indicating the coꢀlocalization of synthesized derivatives into mitochondria in C6 or U87 cells.
5
amphiphilic berberine analogs retain the delocalized
cationiccharge centers that are crucial to selective mitochondrial
18
20 High generation of reactive oxygen species
accumulation. Secondly, it has been verified that the driving
force for such selective accumulationis the mitochondrial
transmembrane potential, which proves to be elevated in cancer
It is well established that reactive oxygen species are implicated
in both cytotoxicity and cancer cell metastasis. Now that
mitochondria are the main sources of intracellular ROS and the
synthesized derivatives seem to preferentially locate to
1
0
30
cells, further favoring the mitochondrial localization propensity.
In addition, yellow fluorescence intensities of 3b, 3c, 5g and 5h
are stronger than that of berberine in both tumor cell lines. The
possible underlying mechanism for the enhanced fluorescence
intensities may be that an increase in the lipophilicity from the
alkyl substituents promotes thepenetration of the synthetic
analogs through cytomembrane, thus improving mitochondrial
25
mitochondria, ROS production of C6 and U87 tumor cells after
incubation with or without compounds 1, 3a-e and 5f-j was
measured by DHE assay. Cellꢀpermeable DHE is oxidized by O2
to ethidium bromide, intercalating the cells’ DNA and staining
the nuclei a fluorescent red that reflects ROS levels. In agreement
1
5
ꢀ
26b
accumulation.
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5
10
Table 2. Physical properties and prediction of BBB penetration of 1, 3a, 3b, 5f, and 5g.
a
b
Calculation
MW
Clog P
0.20
HBA
HBD
PSA
40.82
40.82
40.82
40.82
40.82
Log BB
ꢀ 0.44
0.23
1
371.87
542.51
570.56
603.54
631.59
4
4
4
4
4
0
0
0
0
0
3
3
a
4.665
5.675
5.019
6.029
b
0.39
5
f
0.28
5
g
0.44
>
0.3 (readily cross the BBB);< ꢀ 1.0(only
poorly distributed to the brain)
Lipinski's rules
≤ 500
≤ 5.0
≤ 10
≤ 5
≤ 90
a
MW: molecular weight; clogP: calculated logarithm of the octanolꢀwater partition coefficient; HBA: hydrogenꢀbond acceptor atoms; HBD: hydrogenꢀ
b
bond donor atoms； PSA: polar surface area; compounds with logBB > 0.3 are able to cross the BBB readily, with logBB < ꢀ 1.0 are only poorly
distributed to the brain.
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pathways responsible for cancer cell proliferation, migration and
invasion, the mitochondrionꢀspecific ability and high ROS
production tend to be a tentative underlying mechanism for that
certain derivatives such as 3b, 3c, 5g and 5h possess
antiproliferation, antiꢀmigration and antiꢀinvasion activity at
different dose levels spontaneously. To further understand the
underlying mechanism by which ROS production induced by
berberine derivatives regulates C6 and U87 glioma cells, studies
2
5
30
about the mitochondrial membrane potential(ꢂΨ ), release of
m
cytochrome
C
and apoptosisꢀinducing factor (AIF) and
permeabilization of lysosomes are underway.
35
Improved BBB penetration of berberine derivatives
To target gliomas, BBB penetration is necessary for these
berberine derivatives. As shown in Table 2, berberine only
displayed limited BBB penetration potential, while compounds
3
a and 5g could readily cross the BBB. Therefore, compounds 3a
40
and 5g fulfilthe necessary drugꢀlike and brain penetration criteria.
There results demonstrate that introducing the dodecyl to the Cꢀ9ꢀ
Oꢀposition or Cꢀ13ꢀposition of the berberine scaffold will notably
enhance the BBB penetration of synthetic derivatives, favouring
their antiꢀglioma effects.
45
Conclusions
Glioblastoma still poses an urgent challenge to both patients and
neuroꢀoncologists. Unfortunately, management of glioblastoma is
mainly palliative all over the world. In this study, an effort was
made to develop therapeutics against malignant gliomas through
enhancing the lipophilicity of berberine and retaining the
mitochondriotropic activity, potentially influencing the
intersection of multiple signaling cascades in cancer pathology.
Inspired by our previously evaluated 9ꢀOꢀsubstitued berberine
Figure 7. Reactive oxygen species (ROS) generation induced by
2
ꢁM of compounds 1, 3a-e and 5f-j (n=4). Data are presented as
mean ± standard deviation. A: C6 cells; B: U87 cells. ROS levels
were determined by detection of the dihydroethidium (DHE) red
fluorescence in the nuclei resulting from the oxidation of DHE by
intracellular ROS. P < 0.05; *versus control; **versus berberine.
Con= Control group.
5
50
22
derivatives on C6 glioma cells, 9ꢀOꢀdecylꢀberberine (3a)and
five cationic Cꢀ13ꢀposition berberine derivatives (5f-j) varying in
the length of substituted alkyl chains were synthesized. The data
of biological experiments on C6 and U87 cells suggested that
introduction of the lipophilic substituents with moderate alkyl
chain length to the Cꢀ9ꢀOꢀposition and Cꢀ13ꢀposition of the
55
1
1
2
0
5
0
with the varying patterns of lipophilicity (Table 1) and
cytotoxicity (Figure 1), ROS production of both cell lines after
incubation with1, 3a-e and 5f-j presented similar changing trends
(Figure 7). For instance, those derivatives (3a, 3b, 5f and 5g)
with alkyl chains of moderate length caused significantly higher 60 berberine scaffold led to the identification of several potent
ROS generation than that of compounds 1, 3d, 3e, 5i and 5j,
whose substituents were either longer or shorter alkyl chains or
the benzyl at the Cꢀ9ꢀOꢀ or Cꢀ13ꢀposition on the berberine
scaffold. Combined with the mitochondriotropic attribute, these
proliferation, migration and invasion inhibitors in two
gliomablastoma cell lines. Indeed, the IC50 values for
antiproliferation of several synthesized compounds reached low
micromolar range (3b, 1.24 µM; 5f, 1.12 µM; C6 cells). Over 60%
data indicated that the cytotoxicity displayed by the synthesized 65 of invasion and migration was blocked by the derivatives with
analogues was derived from the induction of high ROS
generation from the mitochondrial organelle.
moderate alkyl chain length (3b and 5g). Further, the
simultaneous inhibitory effects against glioma cell survival,
migration and invasion should be an integrated result of the
improved lipophilicity, the preferential localization into
Because mitochondria are the junction of multiple signal
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neoplastic mitochondria and elevated intracellular ROS
production. Overall, these compounds provide novel
chemotherapeutic candidates and also offer new sight into the
antiglioma therapy through the mitochondrial pathway.
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