Mitochondria-Targeted Lupane Triterpenoid Derivatives and Their Selective Apoptosis-Inducing Anticancer Mechanisms

Article abstract of DOI:10.1021/acs.jmedchem.7b00679

Betulin and betulinic acid have been widely studied for their anticancer activities. However, their further development is limited due to low bioavailability, poor aqueous solubility, and limited intracellular accumulation. In the present study, a triphenylphosphonium cation moiety was linked to betulin and betulinic acid to specifically target them to cancer cell mitochondria. Biological characterization established that uptake of mitochondria-targeted compound 1a in the mitochondria of cancer cells was increased compared to betulin. The mitochondria-targeted derivatives of betulin and betulinic acid showed stronger cytotoxicity than their parent drugs and exhibited more cytotoxic effects in cancer cells than normal cells. The mechanisms may involve the mitochondrial apoptotic pathway, probably caused by the induction of reactive oxygen species production and reducing mitochondrial membrane potential. More importantly, 1a significantly inhibited cancer cell proliferation and migration in an in vivo zebrafish xenograft model. Collectively, these results encourage further study of 1a analogs as anticancer agents.

Full text of DOI:10.1021/acs.jmedchem.7b00679

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Article
Mitochondria-targeted lupane triterpenoid derivatives and
their selective apoptosis-inducing anticancer mechanisms
Yaqing Ye, Tao Zhang, Huiqing Yuan, Defeng Li, Hong-Xiang Lou, and Peihong Fan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00679 • Publication Date (Web): 03 Jul 2017
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Mitochondriaꢀtargeted lupane triterpenoid derivatives and
their selective apoptosisꢀinducing anticancer mechanisms
a
b,d
c
d
a
Yaqing Ye , Tao Zhang , Huiqing Yuan , Defeng Li , Hongxiang Lou ,
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a,
Peihong Fan *
a
Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of
Education, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P.R. China
b
Department of Medicinal Chemistry, Key Lab of Chemical Biology of Ministry of Education,
School of Pharmaceutical Sciences, Shandong University, Jinan 250012, P.R. China
c
Department of Biochemistry & Molecular Biology, School of Medicine, Shandong University,
Jinan 250100, P.R. China
d
Shandong Qidu Pharmaceutical Co. Ltd., Shandong Provincial Key Laboratory of
Neuroprotective Drugs, Zibo 255400, P.R. China
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ABSTRACT: Betulin and betulinic acid have been widely studied for their anticancer
activities. However, their further development is limited due to low bioavailability,
poor aqueous solubility, and limited intracellular accumulation. In the present study, a
triphenylphosphonium cation moiety was linked to betulin and betulinic acid to
specifically target them to cancer cell mitochondria. Biological characterization
established that uptake of mitochondriaꢀtargeted compound 1a in the mitochondria of
cancer cells was increased compared to betulin. The mitochondriaꢀtargeted derivatives
of betulin and betulinic acid showed stronger cytotoxicity than their parent drugs and
exhibited more cytotoxic effects in cancer cells than normal cells. The mechanisms
may involve the mitochondrial apoptotic pathway, probably caused by the induction
of reactive oxygen species production and reducing mitochondrial membrane
potential. More importantly, 1a significantly inhibited cancer cell proliferation and
migration in an in vivo zebrafish xenograft model. Collectively, these results
encourage further study of 1a analogs as anticancer agents.
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Keywords:
anticancer;
mitochondriaꢀtargeted;
betulin;
betulinic
acid;
triphenylphosphonium; apoptosis
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INTRODUCTION
One of the major challenges in developing cancer chemotherapeutics is to improve
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selectivity and to reduce severe toxic side effects for normal cells and tissues. Since
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tumor cells easily gain resistance to apoptosis and mitochondria are tightly associated
2,3
with both intrinsic and extrinsic apoptotic pathways, a promising strategy is to
target therapeutic agents to the mitochondria of cancer cells to improve selectivity and
4,5
the toxicity profile. Furthermore, recent evidence of the mitochondrial membrane
potential (ꢁψ
m
) difference between normal and cancer cells has provided further
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confidence for the strategy of mitochondria targeting in drug design. This strategy
was exemplified by employment of localized lipophilic cations (DLCs) to target
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parent compounds to the mitochondrial matrix. The most studied DLC is the
+
triphenylphosphonium cation (TPP ) and many successful examples have been
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reported with improved antitumor effect and selectivity, e.g. chlorambucil, gallic
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11
acid, and ascorbate, among others.
Recent studies have revealed a wide range of pharmacological activities, including the
antitumor activity of pentacyclic triterpene compounds, such as boswellic acid,
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oleanolic acid, betulinic acid and betulin.
Betulinic acid and betulin are naturally
occurring triterpenes found in birch bark, and studies have shown that betulinic acid
14,15
and betulin exert cytotoxic effects on a variety of malignant tumor cells.
However,
the high hydrophobicity of these natural products hampers their further development
as cytotoxic drugs. A number of studies were attempted by structural modifications to
16–18
improve bioavailability, cytotoxicity and selectivity.
Mechanistic studies
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suggested that the antitumor activity of betulinic acid and betulin is associated with
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the mitochondrial apoptotic pathway by ꢁψ depolarization and the release of
cytochrome c and Smac protein from the mitochondrial membrane. This consequently
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results in activation of caspaseꢀ9, caspaseꢀ3, and caspaseꢀ7 as well as cleavage of poly
14,19,20
(ADPꢀribose) polymerase (PARP), ultimately leading to apoptosis.
Thus, we
could hypothesize that specifically targeting betulinic acid and betulin to cancer cell
mitochondria would improve their cytotoxicity and selectivity. In the present study,
we attempted to transport betulinic acid and betulin to the mitochondria by
+
conjugating them to TPP to improve their antitumor effect. Spivak et al. have
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prepared some TPP derivatives of betulinic acid and evaluated their antitumor
18,21
properties.
Their studies demonstrated that those analogs markedly enhanced the
cytotoxic action as compared to betulinic acid, thus serving as a proof of concept for
such a strategy, and indicated the potential of new cationic derivatives of betulinic
acid. The structural modifications of their compounds are mainly focused on positions
2 or 30 of the betulinic acid skeleton structure via an alkyl chain. Such modifications
may potentially influence the overall activity of analogs since the structural skeleton
was modified. Unlike these biochemically stable linkages using carbonꢀcarbon bonds,
conjugates with ester bonds can be easily hydrolyzed in the cell under the action of
endogenous esterases and have other biokinetic characteristics. Spivak et al. also
reported the potential of linking a TPP cation to a 28ꢀCOOH group of betulinic acid
1
8
by an ester bond . However, continuing to design analogs with an ester linkage at the
OH groups of positions of Cꢀ28 and Cꢀ3 of betulin or betulinic acid can explore the
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+
optimal positions to incorporate the TPP moiety. Such analogs may also provide
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benefits to gain insight into the roles of only mitochondria targeting by the TPP
moiety, since the scaffold of betulin and betulinic acid remain untouched. Herein we
report the design, synthesis, and biological characterization of a series of novel
mitochondriaꢀtargeted derivatives of betulin and betulinic acid with an ester linkage at
the OH groups of the structures to evaluate their mitochondrial uptake and selectivity
by cancer cells, and to test the anticancer activities of these analogs. We also report
the preliminary mechanistic studies of these analogs for observed cytotoxic effects.
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RESULTS
Molecular design and chemical synthesis
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As shown in Figure 1, a TPP moiety was introduced to betulin (1) and betulinic acid
(
2) by an ester linkage. For betulin analogs, the OH group at the Cꢀ28 position was
utilized to incorporate the ester linkage, since the reactivity of the hydroxyl group at
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Cꢀ28 is much higher than that of the Cꢀ3 one. For betulinic acid analogs, the TPP
moiety was linked at the Cꢀ3 OH group. The chemical synthesis of these analogs is
fairly straightforward by esterification of betulin or betulinic acid with
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ωꢀbromoalkanoic acid following standard conditions. As these agents’ anticancer
17
activities can be influenced by the introduction of substituents, we further designed
and synthesized analogs by modification of the remaining hydroxyls. The purity of
target compounds was confirmed by highꢀperformance liquid chromatography (HPLC)
to be ≥95%.
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Figure 1. Chemical structures of the mitoꢀderivatives of betulin (1) and betulinic acid
(2).
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Cytotoxic effects of the mitoꢀderivatives of betulin and betulinic acid.
Table 1. Cytotoxic effects of the mitoꢀderivatives on K562, HLꢀ60, ECAꢀ109, A549 and
a
HepG2 cancer cell lines.
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IC50 (ꢂM)
Compound
K562
HLꢀ60
ECAꢀ109
26.64±2.63
A549
HepG2
HLꢀ7702
26.82±2.25
1
12.89±1.44
30.94±0.52
18.15±0.46
10.70±0.06
b
b
b
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1a
0.57±0.03
0.60±0.03
0.78±0.08
0.61±0.07
1.09±0.05
6.62±0.52
b
b
b
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0.81±0.07
0.71±0.06
0.95±0.12
1.10±0.03
1.11±0.048
8.32±0.36
b
b
b
b
b
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c
0.62±0.06
0.58±0.05
1.41±0.07
0.83±0.20
2.42±0.46
7.18±2.42
e
>200
118.13±3.0
18.47±1.07
111.73±6.3
28.07±3.28
172.21±28.8 158.47±25.1
>200
21.96±1.15
17.13±0.25
25.30±3.81
35.07±3.12
c
c
c
c
c
c
a
b
c
1.24±0.12
0.32±0.09
0.92±0.11
1.26±0.12
1.07±0.02
4.92±0.92
c,d
c,d
c,d
c,d
c,d
c
3.58±0.41
4.03±0.91
3.11±0.49
2.75±0.21
4.75±0.13
4.99±0.57
4.79±0.05
4.35±0.39
4.54±0.38
6.30±1.11
c,d
c,d
c,d
c,d
c,d
c
2
6.04±0.26
7.85±1.65
a
b
The results are expressed as the mean ± standard deviation (SD). P<0.0001 compared to 1.
c
d
e
P<0.0001 compared to 2. P<0.001 compared to 2a. 4ꢀcarboxybutylꢀ triphenylphosphonium
bromide (9) was used as a control.
After chemical synthesis, the designed analogs were evaluated for their cytotoxic
effects against five human cancer cell lines (K562, HLꢀ60, ECAꢀ109, A549 and
HepG2). A normal human hepatocyte cell line (HLꢀ7702) was included to compare
the selectivity of these analogs. As shown in Table 1, the mitoꢀderivatives exhibited
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improved cytotoxic potency (low or subꢀmicromolar potency, and ~5–58ꢀfold
increase) against all five human cancer cell lines compared to their parent compounds
betulin and betulinic acid, respectively. To rule out the possibility that the observed
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ꢀcarboxybutylꢀtriphenylphosphonium bromide (9) was included as control. As can be
seen from Table 1, no cytotoxicity was observed for 9 in any of the tested cancer cells,
thus confirming the essential role of the whole structure of the mitoꢀderivatives.
Although the mitoꢀderivatives showed increased cytotoxic effects towards the normal
HLꢀ7702 cells compared to their parent compounds, comparisons of the IC values
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between tumor line HepG2 cells and HLꢀ7702 cells, these analogs are more cytotoxic
to tumor cells than normal cells, exhibiting some selectivity to cancer cells. No
significant change on the potency of cytotoxicity was observed for analogs with
modification of the Cꢀ3 OH group of 1a, as evidenced by 1b and 1c. Nevertheless, a
slight increase in the IC50 values (up to ~6ꢀfold) was observed when blocking the
hydroxyl of the carboxyl group of 2a.
Among the tested cancer cell lines, the leukemia cells K562 and HLꢀ60 were
relatively more sensitive to the lethal effects of the new analogs. Moreover, 1a and 2a
exhibited the most potent cytotoxic effects compared to other analogs. Therefore, 1a
and 2a were selected for the following pharmacological studies in K562 cells.
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Cellular uptake of 1a in the mitochondria of K562 cells
To confirm that the improved cytotoxicity of the mitoꢀderivatives was a result of
mitochondria targeting, the uptake of 1a and 1 to the mitochondria was evaluated in
K562 cells. As shown in Figure 2, the mitochondrial uptake of 1a was increased
3.4ꢀfold when compared to that of betulin after treatment of K562 cells for 4 h. The
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results demonstrated that the TPP moiety effectively delivered betulin to the
mitochondria.
Figure 2. Cellular uptake of 1a and 1 to the mitochondria of K562 cells. K562 cells were treated
with 10 ꢂM 1a or 1 for 4 h, then mitochondria were isolated and 1a and 1 were extracted with
organic solvent for analysis. (A) RPꢀHPLC chromatograms of 1a standards and of extracts from
the mitochondria of K562 cells. (B) RPꢀHPLC chromatograms of 1 and of extracts from the
mitochondria of K562 cells. (C) Quantitative data of 1a and 1 in mitochondria after normalization
to the protein content. The values are presented as the mean ± SD (n=3). * P<0.01.
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Figure 3. Apoptotic effects of 1a and 2a on K562 cells. (A) K562 cells were treated with the
indicated compounds at indicated concentrations for 48 h. Apoptotic effects were measured by
flow cytometry using Annexin VꢀFITC/PI staining protocol (upper figure for 1a and lower figure
for 2a). (B) Representative histograms for the numbers of cells (% of total) in the early and late
stages of apoptosis for the control and treatment groups. The values are presented as the mean ±
SD (n=3). *P<0.01 compared to control.
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To examine whether the observed cytotoxicity of 1a and 2a is due to induction of
apoptosis, an Annexin VꢀFITC/PI dual staining assay was performed after treatment
of K562 cells. As shown in Figure 3, after 48 h of treatment, significant apoptotic
effects were observed for both 1a and 2a in K562 cells. The analysis of the apoptotic
effects in Figure 3B clearly indicated a doseꢀdependent manner, especially for late
apoptosis. At the maximal tested concentration, 1a (1.2 ꢂM) caused early and late
apoptosis in 12.86% and 38.18% of K562 cells, respectively, and 2a (3.0 ꢂM) caused
early and late apoptosis in 12.62% and 29.77% of K562 cells, respectively. The
results suggested that the observed cytotoxicity of 1a and 2a in cancer cells is mainly
through the apoptotic pathway.
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m
Effects on the mitochondrial membrane potential (ꢁψ ) by mitoꢀderivatives
m
Changes in ꢁψ have been shown to play a significant role in both extrinsic and
2
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intrinsic apoptosis. Since the new analogs were designed to target the mitochondria,
we therefore decided to examine the influence of 1a and 2a on the ꢁψ of K562 cells
m
with the JCꢀ1 staining method. As shown in Figure 4A, treatment of K562 cells with
1
m
a caused significant loss of ꢁψ as reflected by the increase of Jꢀmonomers
(showing green fluorescence and indicating depolarized mitochondria) and concurrent
decrease of Jꢀaggregates (showing red fluorescence and indicating hyperpolarized
mitochondria). The loss of ꢁψ was also confirmed by flow cytometry analysis
m
(Figure 4B) as the intensity of the red fluorescence decreased in a doseꢀdependent
manner after treatment with both compounds.
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Figure 4. Effects on ꢁψ
m
by 1a and 2a in K562 cells. K562 cells were treated with 1a or 2a at the
indicated concentrations for 48 h, and then analyzed by fluorescence microscopy and flow
cytometry after JCꢀ1 staining. (A) Representative fluorescence microcopy images of K562 cells
treated with 1a. The change of ꢁψ
m
was reflected by the disappearance of redꢀstained
m m
mitochondria (negative ꢁψ ) and an increase in greenꢀstained mitochondria (loss of ꢁψ ). (B)
Flow cytometry analysis of K562 cells treated with 1a (upper figure) or 2a (lower figure), along
with the quantification of % of cells with red aggregates. The values are presented as mean ± SD
(
n=3). *P<0.01 compared to control.
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Induction of the production of intracellular reactive oxygen species (ROS)
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Figure 5. Effects of 1a and 2a on ROS production. K562 cells were treated with 1a (A) or 2a (B)
for 48 h, then DCFHꢀDA (10 ꢂM) was loaded and cells were analyzed by flow cytometry
quantification of mitoꢀderivativeꢀinduced oxidative stress in K562 cells. ROSup was tested as a
positive control. The values are presented as the mean ± SD (n=3). *P<0.01 compared to control.
Mitochondria are the main organelle that produces ROS, and ROS has been indicated
24
a double sword role in the cytotoxicity in cancer cells. Therefore, we next evaluated
how treatment with mitoꢀderivatives in K562 cells impact the production of ROS by
flow cytometry analysis. The reagent ROSup from the ROS assay kit was used as a
positive control. Both 1a and 2a induced ROS production at the tested concentrations
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(
0.5 and/or 1.0 µM) (Figure 5). 1a at 0.5 µM or 2a at 1.0 µM increased the ROS level
by approximately 50% compared to the control group.
Mechanistic studies of the apoptotic effects induced by mitoꢀderivatives
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To confirm that the observed apoptotic effects in K562 cells by the mitoꢀderivatives
were exerted via the mitochondrial pathway, we measured the expression levels of
cytochrome c, caspaseꢀ9, cleaved caspaseꢀ9, caspaseꢀ3, cleaved caspaseꢀ3, PARP, and
cleaved PARP using western blotting. As shown in Figure 6, the content of
cytochrome c in the mitochondria decreased, whereas its amount in the cytoplasm
increased, indicating that treatment with the mitoꢀderivatives resulted in loss of ꢁψm
and an increase in the release of cytochrome c from the mitochondria into the cytosol.
Consequently, cytochrome c initiated the activation of caspaseꢀ9, which further
caused an increase in the quantity of cleaved fragments of the downstream effector
caspaseꢀ3. As the end point of this signaling pathway, cleaved PARP was
significantly upꢀregulated and executed the apoptosis program. Treatment with 1a at
4
.0 ꢂM for 48 h dramatically increased the activities of caspaseꢀ9 and caspaseꢀ3, with
.5ꢀ and 2.1ꢀfold increases, respectively, compared with the control.
1
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Figure 6. Effects of 1a and 2a on cytochrome c, caspaseꢀ9, caspaseꢀ3 and PARP. K562 cells were
treated with indicated compounds at indicated concentrations for 48 h. Cells were lysed and the
cell lysates were analyzed by western blotting using the corresponding antibodies. (A)
Representative image of western blotting analysis of cytochrome c in the cytoplasm or
mitochondria. (B) Representative image of western blotting analysis of caspaseꢀ9, cleaved
caspaseꢀ9, caspaseꢀ3, cleaved caspaseꢀ3, PARP and cleaved PARP. (C–D) Quantitative analysis of
western blotting from A and B by Image Lab program with βꢀActin as the internal control. The
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values are presented as the mean ± SD (n=3). Statistical analysis of protein expression,
significantly different from the control *P<0.01.
In vivo activity of mitoꢀderivatives on the growth and metastasis of K562 cells in
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zebrafish
To confirm in vivo activity, 1a was assayed for its effects on the proliferation and
metastasis of K562 cells in zebrafish, a wellꢀstudied and adopted in vivo model that is
2
5,26
widely used for drug screening purposes.
red) were microinjected into zebrafish embryos, and different concentrations of 1a or
were added. Taxol was used as a positive control. As shown in Figure 7A, 48 h after
Specifically, CMꢀDilꢀlabeled K562 cells
(
1
xenotransplantation, the K562 cells in the control group widely migrated away from
the primary site. Notably, the K562ꢀcell xenografts treated with taxol (10.0 ꢂg/mL) or
1
a (1.0 ꢂg/mL, 2.0 ꢂg/mL) (Figure 7A: b, d, and e) exhibited significantly reduced
cell migration. Moreover, the K562ꢀcell xenografts treated with taxol or 1a showed
reduced fluorescence intensities than the control group (Figure 7C), indicating that 1a
could inhibit the growth of K562 cells in zebrafish xenografts. It is worth noting that
1
a effectively inhibited the proliferation and metastasis of K562 cells in zebrafish
xenografts at a low concentration (1.0 ꢂg/mL), which was 1/10 the concentration of
the positive control, taxol, and that the in vivo activity of 1a was obviously increased
compared with that of betulin.
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Figure 7. Inhibitory effect of 1a on the proliferation and metastasis of K562 cells in zebrafish
xenografts. CMꢀDilꢀlabeled K562 cells (red) were microinjected into zebrafish embryos, and
different concentrations of 1a or 1 were added. Taxol was used as a positive control. After 48 h,
the proliferation and metastasis of the xenografts of K562 cells were imaged under a confocal
microscope. (A) Metastasis of K562ꢀcell xenografts. (B) Quantification of the fluorescent area of
the tumor xenografts, representing K562 cell metastasis. (C) Fluorescence intensity of the tumor
xenografts, representing the number of K562 cells. The results are presented as the mean ± SD
(
n=15). *P<0.01 compared to control.
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DISCUSSION AND CONCLUSIONS
The triterpenoids betulin and betulinic acid have attracted extensive attention due to
their broadꢀspectrum biological activities, and as potential anticancer treatments.
Analogs based on the structures of betulin and betulinic acid have been designed and
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developed to improve antitumor activity and pharmacokinetic properties.
Although promising new analogs were identified with improved therapeutic potential,
more effort is required to improve activity and selectivity and to identify their precise
mechanisms of action.
Cancer cells commonly exhibit elevated ꢁψ of at least 60 mV compared to normal
m
28
cells. The difference of ꢁψ
m
between cancer cells and normal cells could be
exploited therapeutically to selectively target compounds to the mitochondria of
cancer cells. DLCs can easily penetrate the lipid bilayer due to the even distribution of
charge over a large hydrophobic surface area, thereby lowering the activation energy
4
required for cation movement across the membrane. According to the Nernst
equation, an approximate 10ꢀfold increase of mitochondrial uptake should be
2
9
expected for a cation for every 60 mV increase in ꢁψ
m
.
m
Generally, ꢁψ is
approximately 180–200 mV, the maximum value that a bilayer can sustain while
2
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maintaining its integrity.
The wellꢀcharacterized and most widely used DLC for
+
29
mitochondria targeting is the TPP cation. Proofꢀofꢀconcept studies for utilizing
+
TPP as a mitochondriaꢀtargeting moiety have been achieved by in vivo studies of
8
,10,32
novel mitoꢀderivatives in animal models of human cancer.
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In the present study, based on the hypothesis that the cytotoxicity of a given
compound to cancer cells is closely associated with its accumulation in the
mitochondria, we designed and synthesized six TPPꢀlinked derivatives of betulin and
betulinic acid and compared their biological activity with betulin and betulinic acid.
Our results revealed that the antiꢀproliferative effects of the mitoꢀderivatives are
significantly improved compared to the parent compounds against all tested tumor
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cells (Table 1). In contrast, a simple TPP derivative alone exerted no cytotoxicity to
either cancer cells or normal cells, confirming the essential role of the whole structure
of the designed compounds. Moreover, the lower toxicities of these mitoꢀderivatives
to the normal hepatocyte cell line HLꢀ7702 compared with hepatoma carcinoma cells
were observed. A similar effect was observed in a recent study of delocalized
8
lipophilic cations of gallic acid derivatives. The tumor cellꢀselective cytotoxicity of
these derivatives could be further explored by testing more cell lines in the future. The
results also demonstrated that the OH groups of the positions of Cꢀ28 of betulin and
+
Cꢀ3 of betulinic acid were optional positions to incorporate the TPP moiety. This
design left the scaffold of betulin and betulinic acid untouched, but improved
cytotoxicity significantly, supporting the important role of mitochondria targeting by
+
the TPP moiety. Importantly, another advantage of such a design is the simple
synthetic procedure with an ester linkage.
Our studies also suggested that masking the 3ꢀOH group of betulin with an ester
moiety did not significantly change the cytotoxic potency or selectivity to cancer cells.
+
This may serve as indirect evidence to support the essential role of the TPP moiety in
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mitochondria uptake. A similar effect was observed in a recent study of resveratrol
analogs showing that acetylated or free hydroxyl analogs exhibited comparable
33
cytotoxicity. Interestingly, it was reported that alkylation of the 28ꢀCOOH group of
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betulinic acid led to the loss of its antitumor activity. Here, when the carboxyl group
of the Cꢀ28 position of betulinic acid was masked with an ester, the potency was
decreased compared to the free carboxylic group 2a, but they are much more potent
than betulinic acid. However, the decrease of potency when masking the Cꢀ28
position might suggest that the Cꢀ3 OH could be a better position for incorporation of
+
the TPP moiety than Cꢀ28 COOH, but more studies are warranted to further support
this.
m
It has been reported that ROS induction and change of ꢁψ trigger the release of
3
4
cytochrome c release, and subsequently apoptosis through caspaseꢀ3 activation. Our
mechanistic study results are consistent with this by showing that treatment of K562
cells with mitoꢀderivatives induced significant production of ROS and reduced ꢁψm.
The activation of caspaseꢀ9 and caspaseꢀ3 as well as the cleavage of PARP were
observed under the same experimental conditions. In addition, the level of
cytochrome c in the mitochondria was decreased, an event accompanied by an
increase of cytosolic level. The results are also consistent with previous studies on the
anticancer mechanisms of betulin and betulinic acid. Notably, treatment of zebrafish
with 1a exhibited significant inhibition on the xenograft proliferation and migration of
K562 cells, thus confirming the in vivo activity of 1a. In summary, our studies
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provide further evidence that natural pentacyclic triterpene compounds can be
optimized by the mitochondriaꢀtargeting strategy to improve potency and selectivity.
EXPERIMENTAL SECTION
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1
13
Chemistry. We recorded the H and C NMR spectra with a Bruker AV
1
13
spectrometer operating at 400 ( H) and 100 ( C) MHz or a Bruker Avance DRXꢀ600
1
13
spectrometer operating at 600 ( H) and 150 ( C) MHz, with tetramethylsilane (TMS)
as an internal standard. The chemical shifts are reported as δ (ppm) downfield from
1
TMS for H NMR. Column chromatography (CC) was performed with silica gel
(
200−300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). TLC for
monitoring was performed with precoated silica gel GFꢀ254 glass plates (Qingdao
Haiyang Chemical Co. Ltd., Qingdao, China). betulin and betulinic acid were
purchased from the Baoji Herbest BioꢀTech Co. Ltd., Shaanxi, China. All other
organic compounds, inorganic salts, acids, and solvents were purchased from the
Shanghai Xianding Biological Science & Technology Co. Ltd., China. All final
compounds were analyzed for purity by highꢀperformance liquid chromatography
(
HPLC) and were found to have ≥95% purity. HPLC analysis was performed on an
Agilent 1100 series with a ZORBAX SBꢀC18 column (250 mm × 4.6 mm, 5 ꢂm). The
solvent for compounds was 0.1% formic acid in acetonitrile, and eluent was 90%
acetonitrile/water. The absorbance was detected at 220 nm.
3
βꢀHydroxylupꢀ20(29)ꢀenꢀ28ꢀyl 5ꢀ(triphenylphosphonio)pentanoate bromide (1a).
Triphenylphosphine (325 mg, 1.24 mmol) was added to a solution of
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βꢀhydroxylupꢀ20(29)ꢀenꢀ28ꢀyl 5ꢀbromopentanoate (intermediate 3, 200 mg, 0.33
3
mmol; for structure, see supporting file) in CH CN (10 mL). The mixture was then
stirred at 80°C until the reaction was complete according to TLC detection. The
solvent was subsequently removed under diminished pressure, and the residue was
purified by CC using (30:1→20:1) DCM/MeOH to obtain 30 mg (15%) of 1a as a
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D
1
white solid. Mp: 124–129°C; [α]
MHz): δ 7.68–7.89 (m, 15H, PhꢀH), 4.67 (s, 1H, Hꢀ29a), 4.58 (s, 1H, Hꢀ29b), 4.18 (d,
J = 11.0 Hz, 1H, Hꢀ28a), 3.96–3.95 (m, 2H, CH ꢀPꢀ), 3.77 (d, J = 11.0 Hz, 1H,
3 3
= +3.6 (c 0.1, CH OH); H NMR (CDCl , 600
2
Hꢀ28b), 3.18 (dd, J = 11.5, 4.7 Hz, 1H, Hꢀ3), 2.42 (t, J = 6.8 Hz, 2H, ꢀCOCH2),
2
2
.41–2.36 (m, 1H, Hꢀ19), 2.04–0.90 (m, 28H, СН, СН in pentacyclic skeleton or
3
carbon chain), 1.66, 1.00, 0.97, 0.95, 0.82, 0.76 (each, s, 3H, CH ꢀ23, 24, 25, 26, 27,
13
3
0); C NMR (CDCl
3
, 150 MHz): δ 173.6 (OꢀCOꢀ), 150.1 (Cꢀ20), 134.9 (d, JC,P = 3.0
Hz, PhꢀC), 133.8 (d, JC,P = 10.0 Hz, PhꢀC), 130.5 (d, JC,P = 12.5 Hz, PhꢀC), 118.4 (d,
JC,P = 85.9 Hz, PhꢀC), 109.9 (Cꢀ29), 78.9 (Cꢀ3), 77.3, 77.0, 76.8, 62.5, 55.3, 53.5,
50.3, 48.7, 47.7, 46.3, 42.7, 40.8, 38.8, 38.7, 37.6, 37.1, 34.5, 34.2, 33.5, 29.7, 29.5,
2
8.0, 27.4, 27.0, 25.5, 25.2, 22.8, 22.5, 22.0, 20.8, 19.1, 18.3, 16.0, 15.4, 14.7; HPLC
+
+
72 3
purity: 99%. HRꢀESIꢀMS m/z calculated for C53H O P [MꢀBr] 787.5214, found
7
87.5231.
3
βꢀFormyloxylupꢀ20(29)ꢀenꢀ28ꢀyl 5ꢀ(triphenylphosphonio)pentanoate bromide (1b).
Triphenylphosphine (310 mg, 1.18 mmol) was added to a solution of
βꢀformyloxylupꢀ20(29)ꢀenꢀ28ꢀyl 5ꢀbromopentanoate (intermediate 4, 150 mg, 0.235
mmol; for structure, see supporting file) in CH CN (3 mL). The mixture was then
3
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stirred at 80°C until the reaction was complete according to TLC detection. The
solvent was subsequently removed under diminished pressure, and the residue was
purified by preparative TLC by elution with DCM: MeOH (10:1) to obtain 55 mg
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(
36.7%) of 1b as a yellow solid. Mp: 118–123°C; [α]D = +13.5 (c 0.1, CH OH); H
3
NMR (CD
3
OD, 600 MHz): δ 8.13 (s, 1H, ꢀCHO), 7.89–7.75 (m, 15H, PhꢀH), 4.73 (s,
H, Hꢀ29a), 4.62 (s, 1H, Hꢀ29b), 4.58 (dd, J = 12.0, 6.0 Hz, 1H, Hꢀ3), 4.37 (d, J =
1.1 Hz, 1H, Hꢀ28a), 3.80 (d, J = 11.1 Hz, 1H, Hꢀ28b), 3.45 (t, J = 14.7 Hz, 2H,
ꢀPꢀ), 2.49–2.48 (m, 1H, Hꢀ19), 2.46 (t, J = 6.9 Hz, 2H, ꢀCOCH ꢀ), 1.68, 1.05,
.01, 0.90 (each, s, 3H, CH ꢀ25, 26, 27, 30), 0.88 (s, 6H, CH ꢀ23, 24), 1.91–1.09 (m,
1
1
CH
2
2
1
2
3
3
13
2 3
8H, СН, СН in pentacyclic skeleton or carbon chain); C NMR (CD OD, 150
MHz): δ 173.6 (OꢀCOꢀ), 161.7 (ꢀCHO), 149.9 (Cꢀ20), 134.9 (d, JC,P = 2.9 Hz, PhꢀC),
33.4 (d, JC,P = 10.0 Hz, PhꢀC), 130.2 (d, JC,P = 12.6 Hz, PhꢀC), 118.5 (d, JC,P = 86.4
Hz, PhꢀC), 109.2 (Cꢀ20), 80.9 (Cꢀ3), 62.2 (Cꢀ28), 55.3, 50.2, 48.6, 46.4, 42.4, 40.7,
8.1, 37.6, 37.4, 36.8, 34.2, 33.9, 32.7, 29.4, 29.2, 26.9, 26.8, 25.5, 25.4, 25.1, 23.5,
21.7, 21.6, 21.2, 20.9, 20.5, 17.9, 17.9, 15.6, 15.3, 15.2, 13.8; HPLC purity: 96%.
1
3
+
+
72 4
HRꢀESIꢀMS m/z calculated for C54H O P [MꢀBr] 815.5163, found 815.5170.
3
βꢀAcetyloxylupꢀ20(29)ꢀenꢀ28ꢀyl 5ꢀ(triphenylphosphonio)pentanoate bromide (1c).
Triphenylphosphine (430 mg, 1.66 mmol) was added to a solution of
βꢀacetyloxylupꢀ20(29)ꢀenꢀ28ꢀyl 5ꢀbromopentanoate (intermediate 5, 210 mg, 0.33
mmol; for structure, see supporting file) in CH CN (3 mL). The mixture was then
3
3
stirred at 80°C until the reaction was complete according to TLC detection. The
solvent was subsequently removed under diminished pressure, and the residue was
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purified by preparative TLC by elution with DCM: MeOH (10:1) to obtain 70 mg
2
0
1
(23%) of 1c as a white solid. Mp: 147–156°C; [α]
D
3
= +8.6 (c 0.1, CH OH); H NMR
(
CD OD, 600 MHz): δ 7.93–7.77 (m, 15H, PhꢀH), 4.73 (s, 1H, Hꢀ29a), 4.62 (s, 1H,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
1
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8
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4
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9
0
Hꢀ29b), 4.46 (dd, J = 11.4, 4.9 Hz, 1H, Hꢀ3), 4.40 (d, J = 10.7 Hz, 1H, Hꢀ28a), 3.83
(d, J = 11.1 Hz, 1H, Hꢀ28b), 3.47 (ddd, J = 13.6, 9.4, 7.0 Hz, 2H, CH
2
ꢀPꢀ), 2.49–2.47
(
m, 1H, Hꢀ19), 2.46 (t, J = 7.1 Hz, 2H, ꢀCOCH ꢀ), 2.04 (s, 3H, CH COꢀ), 1.71, 1.07,
2
3
1
.03, 0.92, 0.89, 0.88 (each, s, 3H, CH
3
ꢀ23, 24, 25, 26, 27, 30), 1.91–1.25 (m, 28H,
13
СН, СН
2
in pentacyclic skeleton or carbon chain); C NMR (CD
3
OD, 150 MHz): δ
1
73.5 (OꢀCOꢀ), 171.5 (ꢀCOCH ), 149.9 (Cꢀ20), 135.0 (d, J = 3.0 Hz, PhꢀC), 133.4
3 C,P
(
d, JC,P = 10.0 Hz, PhꢀC), 130.2 (d, JC,P = 12.6 Hz, PhꢀC), 118.5 (d, JC,P = 86.4 Hz,
PhꢀC), 109.2 (Cꢀ29), 81.0 (Cꢀ3), 62.2 (Cꢀ28), 55.4, 50.2, 48.6, 46.4, 42.4, 40.7, 38.1,
7.6, 37.5, 36.8, 34.1, 33.9, 32.7, 29.4, 29.2, 27.1, 26.7, 25.5, 25.4, 25.1, 23.3, 21.7,
21.2, 20.9, 20.5, 19.8, 17.9, 15.6, 15.3, 15.2, 13.8; HPLC purity: 96%. HRꢀESIꢀMS
3
+
+
m/z calculated for C H O P [MꢀBr] 829.5319, found 829.5333.
55
74
4
[
3βꢀ(5ꢀ(Triphenylphosphonio)pentanoyl)oxylupꢀ20(29)ꢀenꢀ28ꢀoic acid] bromide
(2a). Triphenylphosphine (292 mg, 1.11 mmol) was added to a solution of
βꢀ(5ꢀbromopentanoyl)lupꢀ20(29)ꢀenꢀ28ꢀoic acid (intermediate 6, 230 mg, 0.37 mmol;
for structure, see supporting file) in CH CN (10 mL). The mixture was then stirred at
0°C until the reaction was complete according to TLC detection. The solvent was
3
3
8
subsequently removed under diminished pressure, and the residue was purified by
preparative TLC by elution with DCM: MeOH (10:1) to obtain 83 mg (28%) of 2a as
20
D
1
a white solid. Mp: 135–143°C; [α]
3 3
= +15.2 (c 0.1, CH OH); H NMR (CDCl , 400
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MHz): δ 7.88–7.67 (m, 15H, PhꢀH), 4.73 (s, 1H, Hꢀ29a), 4.61 (s, 1H, Hꢀ29b), 4.37 (dd,
J = 10.6, 5.8 Hz, 1H, Hꢀ3), 3.91 (t, J = 14.1 Hz, 2H, CH
Hz, 1H, Hꢀ20), 2.38 (t, J = 6.8 Hz, 2H, ꢀOCOCH ꢀ), 1.69, 0.96, 0.88, 0.81 0.75, 0.72
each, s, 3H, CH ꢀ23, 24, 25, 26, 27, 30), 2.29–1.31 (m, 29H, СН, СН in pentacyclic
2
ꢀPꢀ), 3.01 (td, J = 10.5, 4.5
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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29
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
3
2
1
3
skeleton or carbon chain); C NMR (CDCl
CH COꢀ), 150.5 (Cꢀ20), 135.0 (d, JC,P = 2.9 Hz, PhꢀC), 133.7 (d, JC,P = 10.0 Hz,
PhꢀC), 130.5 (d, JC,P = 12.5 Hz, PhꢀC), 118.8 (d, JC,P = 85.9 Hz, PhꢀC), 109.7 (Cꢀ29),
3
, 100 MHz): δ 181.0 (Cꢀ28), 172.9
(
2
8
0.9 (Cꢀ3), 77.4, 77.0, 76.7, 65.3, 56.3, 55.4, 50.4, 49.3, 46.9, 42.4, 41.9, 40.7, 38.4,
7.8, 37.1, 34.2, 33.9, 32.2, 30.6, 30.1, 29.7, 29.1, 28.0, 25.4, 23.7, 23.3, 23.1, 22.9,
1.9, 20.9, 19.4, 18.1, 16.6, 16.1, 14.6, 14.1; HPLC purity: 97%. HRꢀESIꢀMS m/z
3
2
+
+
70 4
calculated for C53H O P [MꢀBr] 801.5006, found 801.5020.
[Methylꢀ3βꢀ(5ꢀ(triphenylphosphonio)pentanoyl)oxylupꢀ20(29)ꢀenꢀ28ꢀoate] bromide
2b). Triphenylphosphine (376 mg, 1.24 mmol) was added to a solution of
methylꢀ3βꢀ(5ꢀbromopentanoyl)lupꢀ20(29)ꢀenꢀ28ꢀoate (intermediate 7, 180 mg, 0.28
mmol; for structure, see supporting file) in CH CN (10 mL). The mixture was then
(
3
stirred at 80°C until the reaction was complete according to TLC detection. The
solvent was subsequently removed under diminished pressure, and the residue was
purified by preparative TLC by elution with DCM: MeOH (10:1) to obtain 66 mg
2
0
1
(
37%) of 2b as a yellow solid. Mp: 168–177°C; [α]D = +11.3 (c 0.1, CH OH); H
3
NMR (CD
3
OD, 600 MHz): δ 7.90–7.75 (m, 15H, PhꢀH), 4.71 (s, 1H, Hꢀ29a), 4.60 (s,
1
2
H, Hꢀ29b), 4.40 (dd, J = 11.6, 4.9 Hz, 1H, Hꢀ3), 3.65 (s, 3H, CH Oꢀ), 3.49ꢀ3.44 (m,
3
H, CH
2
2
Brꢀ), 3.00 (td, J = 10.9, 5.1 Hz, 1H, Hꢀ19), 2.40 (t, J = 7.1 Hz, 2H, ꢀCH COꢀ),
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6
1
.69, 1.01, 0.94, 0.86, 0.80, 0.77 (each, s, 3H, CH
m, 28H, СН, СН in pentacyclic skeleton or carbon chain); C NMR (CD
MHz): δ 176.2 (Cꢀ28), 173.1 (CH COꢀ), 150.4 (Cꢀ20), 134.9 (d, JC,P = 3.0 Hz, PhꢀC),
33.5 (d, J = 10.0 Hz, PhꢀC), 130.1 (d, J = 11.3 Hz, PhꢀC), 118.5 (d, JC,P = 86.4
3
ꢀ23, 24, 25, 26, 27, 30), 2.27–1.08
13
(
2
3
OD, 150
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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4
5
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0
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2
3
4
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8
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1
C,P
C,P
Hz, PhꢀC), 108.9 (Cꢀ29), 81.1 (Cꢀ3), 59.6, 56.3, 55.4, 50.4, 49.2, 42.2, 40.6, 38.2,
3
8.1, 37.5, 36.9, 36.5, 34.0, 33.1, 31.7, 30.3, 29.4, 27.2, 25.6, 25.5, 25.4, 23.3, 21.7,
1.7, 21.3, 20.9, 20.7, 18.2, 17.8, 15.7, 15.4, 15.2, 13.7, 13.3; HPLC purity: 98%.
2
+
+
72 4
HRꢀESIꢀMS m/z calculated for C54H O P [MꢀBr] 815.5163, found 815.5176.
[Ethylꢀ3βꢀ(5ꢀ(triphenylphosphonio)pentanoyl)oxylupꢀ20(29)ꢀenꢀ28ꢀoate]bromide
(2c). Triphenylphosphine (244 mg, 0.89 mmol) was added to a solution of
ethylꢀ3βꢀ(5ꢀbromopentanoyl)lupꢀ20(29)ꢀenꢀ28ꢀoate (intermediate 8, 115 mg, 0.18
3
mmol; for structure, see supporting file) in CH CN (10 mL). The mixture was then
stirred at 80°C until the reaction was complete according to TLC detection. The
solvent was subsequently removed under diminished pressure, and the residue was
purified by preparative TLC by elution with DCM: MeOH (10:1) to obtain 60 mg
2
D
0
1
(52%) of 2c as a white solid. Mp: 150–154°C; [α]
NMR (CD OD, 600 MHz): δ 7.91–7.75 (m, 15H, PhꢀH), 4.72 (s, 1H, Hꢀ29a), 4.60 (s,
1H, Hꢀ29b), 4.40 (dd, J = 11.6, 4.9 Hz, 1H, Hꢀ3), 4.18–4.10 (m, 2H, CH CH Oꢀ),
3
= +16.2 (c 0.1, CH OH); H
3
3
2
3
.48–3.42 (m, 2H, CH Brꢀ), 3.03–2.99 (td, J = 10.8, 4.8 Hz, 1H, Hꢀ19) 2.40 (t, J = 7.1
2
Hz, 2H, CH
2
COꢀ), 1.26 (t, J = 7.1 Hz, 3H, CH
3
CH
2
Oꢀ) 1.70, 1.02, 0.95, 0.87, 0.80,
in
OD, 150 MHz): δ 176.2 (Cꢀ28),
0
.77 (each, s, 3H, CH
3 2
ꢀ23, 24, 25, 26, 27, 30), 2.29–1.29 (m, 28H, СН, СН
13
pentacyclic skeleton or carbon chain); C NMR (CD
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73.1 (CH
2
COꢀ), 150.4 (Cꢀ20), 135.0 (d, JC,P = 3.0 Hz, PhꢀC), 133.4 (d, JC,P = 10.0
Hz, PhꢀC), 130.2 (d, JC,P = 12.6 Hz, PhꢀC), 118.5 (d, JC,P = 86.4 Hz, PhꢀC), 108.9
Cꢀ29), 81.1(Cꢀ3), 59.6, 56.3, 55.4, 50.4, 49.2, 42.2, 40.6, 38.3, 38.1, 37.5, 36.9, 36.6,
(
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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35
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41
42
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54
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3
4.0, 33.1, 31.7, 30.3, 29.4, 27.2, 25.6, 25.5, 25.4, 23.3, 21.7, 21.7, 21.3, 20.9, 20.7,
1
8.2, 17.9, 15.7, 15.4, 15.2, 13.8, 13.3; HPLC purity: 97%. HRꢀESIꢀMS m/z
+
+
74 4
calculated for C55H O P [MꢀBr] 829.5316, found 829.5332.
Biological study
Cells and Materials. Human chronic myeloid leukemia cells (K562) and human
promyelocytic leukemia cells (HLꢀ60) were purchased from the Type Culture
Collection of the Chinese Academy of Sciences, Shanghai, China. Human alveolar
adenocarcinoma cells (A549), human esophageal squamous carcinoma cells
(
ECAꢀ109), human hepatoblastoma cells (HepG2) and human normal liver cells
HLꢀ7702) were obtained from the School of Pharmaceutical Science of Shandong
(
University. K562 cells, A549 cells, ECAꢀ109 cells, HepG2 cells and HLꢀ7702 cells
were cultured in RPMIꢀ1604 medium (Macgene, Beijing, China), and HLꢀ60 cells
were grown in IMDM (Macgene, Beijing, China). The media were supplemented with
1
0% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 100
U/mL penicillin and 0.1 mg/mL streptomycin (Solarbio, Beijing, China), and the cells
were incubated at 37°C with 5% CO . The Annexin VꢀFITC Apoptosis Detection Kit
2
was obtained from Keygen Biotech. JCꢀ1 and the ROS Assay Kit was from Beyotime
(Nanjing, China). Finally, the primary antibodies against caspaseꢀ3, cleaved caspaseꢀ3,
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PARP, cytochrome c, caspaseꢀ9 and βꢀactin were purchased from Cell Signaling
Technology (Boston, MA, USA).
MTT Assay. Cells were seeded in 96ꢀwell plates at a density of 5000 cells per well.
For the solid tumor lines (A549, ECAꢀ109, HepG2 and HLꢀ7702), after 16–24 h, the
cells were treated with a solution of one of the novel synthesized compounds or with
TPP for 48 h. After the medium was refreshed, MTT solution (5 mg/mL, 10 ꢂL;
Solarbio, Beijing, China) was added to each well, and the cells were incubated with
the MTT for 4 h at 37°C. After the medium was removed, 100 ꢂL of DMSO (Sigma,
St. Louis, MO, USA) was added to each well, and the OD570 was measured using a
microplate reader (BioꢀRad, CA, USA). For hematological cells (K562 and HLꢀ60), at
the end of the drug treatment, 10 ꢂL of MTT solution was added to each well, and the
samples were incubated at 37°C for 4 h. After the plates were centrifuged at 3000 rpm
for 30 min, the medium was removed, 100 ꢂL of DMSO (Sigma, St. Louis, MO, USA)
was added to each well, and the OD570 was measured as described above. The test
results are expressed as the concentration of the test compound that inhibited cell
growth by 50% (IC50). All assays were performed in triplicate.
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Flow cytometry assay. Cell culture. K562 cells at 4×10 cells/well in a final volume
of 2 mL were seeded in 6ꢀwell plates. After 12 h of incubation, various concentrations
of 1a or 2a were added to the wells for various periods of time. Cell apoptosis
analysis. Cells were harvested at 2000 rpm for 5 min and then washed with iceꢀcold
PBS twice, followed by resuspension in binding buffer. Next, the samples were
incubated with 5 ꢂL of Annexin V and 5 ꢂL of PI for 15 min at room temperature in
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the dark. Finally, the cells were analyzed by flow cytometry (Becton Dickinson, NJ,
USA) within 1 h. Mitochondrial membrane potential. Cells were harvested at 2000
rpm for 5 min and then washed twice with iceꢀcold PBS, followed by resuspension in
JCꢀ1 (5 mg/mL) and incubation at 37°C for 15 min. After the cells were rinsed three
times and suspended in PBS, the JCꢀ1 fluorescence was observed by flow cytometry.
Detection of intracellular ROS. Intracellular ROS generation was examined using 2′,
10
11
12
13
14
15
16
17
18
19
20
21
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23
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60
7
2
′ꢀdichlorofluorescein diacetate (DCFHꢀDA). For this purpose, cells were harvested at
000 rpm for 5 min and then washed twice with iceꢀcold PBS, followed by
resuspension in 0.5 mL of FBSꢀfree medium containing 10 mM DCFHꢀDA and
incubation at 37°C for 30 min. After the cells were rinsed three times and suspended
in PBS, ROS production in the cells was immediately monitored by flow cytometry.
Western blotting. Protein extraction. Cells were washed with iceꢀcold PBS and
centrifuged for 5 min at 2000 rpm. The pellets were then resuspended in 100 ꢂL of
Lysis Buffer (Solarbio, Beijing, China) and incubated on ice for 30 min. The cells
were subsequently centrifuged for 10 min at 14,000 rpm, after which the supernatants
were collected as wholeꢀcell lysates. The cytosolic and mitochondrial fractions were
isolated from the treated cells using the Cell Mitochondrial Isolation Kit (Beyotime,
Nanjing, China) following the manufacturer’s recommended protocol. The protein
levels were quantified with a BCA Assay Kit (Beyotime). Western blotting. Protein
samples (60 ꢂg/lane) were subjected to SDSꢀPAGE (BioꢀRad) and electrotransferred
to PVDF membranes (Millipore, MA, USA). The membranes were then blocked with
5
% skim milk in TBST at room temperature for 4 h, followed by three washes with
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