Scutellarin inhibits Hela cell growth and glycolysis by inhibiting the activity of pyruvate kinase M2

Article abstract of DOI:10.1016/j.bmcl.2017.11.011

Scutellarin, one of natural flavonoids, is widely and clinically used for treating many diseases in China. Recently, scutellarin has demonstrated a broad spectrum of anti-proliferative activities against multiple cancer cell lines. However, the molecular mechanism of action remains to be investigated. We herein report the design and synthesis of biotinylated scutellareins as probes, which can be applied to discover scutellarein interacting proteins. Finally, we show that scutellarin directly targets pyruvate kinase M2 (PKM2) and inhibits its cytosolic activity to decrease glycolytic metabolism; on the other hand, scutellarin may also participate in regulating cell cycle and apoptotic proteins by activating MEK/ERK/PIN1 signaling pathway to promote the nuclear translocation of PKM2.

Full text of DOI:10.1016/j.bmcl.2017.11.011

Accepted Manuscript
Scutellarin inhibits Hela cell growth and glycolysis by inhibiting the activity of
pyruvate kinase M2
Lin You, Hong Zhu, Chun Wang, Fang Wang, Yongjun Li, Yan Li, Yonglin
Wang, Bin He
PII:
S0960-894X(17)31088-0
https://doi.org/10.1016/j.bmcl.2017.11.011
BMCL 25417
DOI:
Reference:
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Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
12 September 2017
3 November 2017
6 November 2017
Please cite this article as: You, L., Zhu, H., Wang, C., Wang, F., Li, Y., Li, Y., Wang, Y., He, B., Scutellarin inhibits
Hela cell growth and glycolysis by inhibiting the activity of pyruvate kinase M2, Bioorganic & Medicinal Chemistry
Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.11.011
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Scutellarin inhibits Hela cell growth and glycolysis by inhibiting the activity of
pyruvate kinase M2
a,†,§
a,§
a
a
a
b,*
a,*
Hong Zhu, Chun Wang, Fang Wang, Yongjun Li, Yan Li, Yonglin Wang, Bin He
a,*
Lin You,
a
Engineering Research Center for the Development and Application of Ethnic Medicine and TCM (Ministry of Education), Guizhou Provincial Key Laboratory
of Pharmaceutics, School of Pharmacy, National Engineering Research Center of Miao’s Medicines, Guizhou Medical University, Guiyang, Guizhou 550004,
China.
b
School of Basic Medicine, Guizhou Medical University, Guizyang, Guizhou 550004, China.
†
§
∗
Current address: Shuicheng County People's Hospital，Liupanshui, Guizhou 553000, China.
These authors contributed equally to this work.
Corresponding author. Tel.: +086-851-691-0028; fax: +086-851-691-0028; e-mail: binhe@gmc.edu.cn
ARTICLE INFO
ABSTRACT
Article history:
Received
Scutellarin, one of natural flavonoids, is widely and clinically used for treating many diseases in
China. Recently, scutellarin has demonstrated a broad spectrum of anti-proliferative activities
against multiple cancer cell lines. However, the molecular mechanism of action remains to be
investigated. We herein report the design and synthesis of biotinylated scutellareins as probes,
which can be applied to discover scutellarein interacting proteins. Finally, we show that scutellarin
directly targets pyruvate kinase M2 (PKM2) and inhibits its cytosolic activity to decrease glycolytic
metabolism; on the other hand, may also participate in regulating cell cycle and apoptotic proteins
by activating MEK/ERK/PIN1 signaling pathway to promote the nuclear translocation of PKM2.
Revised
Accepted
Available online
Keywords:
Scutellarin
Scutellarein
PKM2
2009 Elsevier Ltd. All rights reserved.
Inhibitor
Antitumor activity
Traditional Chinese Medicines (TCMs) have been clinically
used in treating many human diseases for a long period, which
are regarded as potential sources for modern drug discovery [1,
Due to the significance of scutellarin in the clinical therapy,
scutellarin has been attracted increasingly attentions in recent
years. Among these, a few reports focus on the study of
scutellarin in treating cancer [6]. For an example, Scutellarin
diminished the proliferation of B-lymphoma Namalwa cells in
vitro and inhibited lymphoma growth in Namalwa cell-
xenotransplanted mice without obvious toxicity [7]. Besides,
scutellarin also showed a widely inhibitory effect on the cell
proliferation of 35 human tumor cell lines with an IC50 (50%
2
]. Flavonoids are found in many TCMs, which are
polyphenolic compounds that have been shown to possess a
variety of biological activities at nontoxic concentrations in
organisms [3]. Scutellarin (or Scutellarein-7-O-glucuronide),
one of flavonoids, a clinic natural drug consisting of total
flavonoids of Erigeron breviscapus (Vant.) Hand-Mazz.
(
Compositae), has been widely used for the treatment of
inhibitory concentration) range of 15.17-78.95 µM [7].
cerebral infarction, coronary heart disease, angina pectoris and
other cardiocerebrovascular diseases in China (Fig 1) [4].
Pharmacological studies have demonstrated that the major
effective form of scutellarin in body is scutellarein, which
readily come from scutellarin, and is easily absorbed into the
blood and then metabolized (Fig 1) [5].
However, there still remain many questions to be answered. For
examples, what’s the mechanism of scutellarin in treating
cancer? And how can we develop better compounds for treating
cancer based on the scaffold of scutellarin? To address these
questions, we should figure out the direct targets of scutellarin
in cancer therapy and further investigate its molecular
mechanism of action. Chemical biology is a powerful tool to
answer above questions. Using this strategy, many successes
have been made in many mechanism studies of natural
products. For artemisinin, a world famous anti-malarial drug,
Wang group disclosed that haem is predominantly responsible
for artemisinin activation in parasite killing [8]. For adenanthin,
a diterpenoid isolated from the leaves of Rabdosia adenantha,
which induces differentiation of acute promyelocytic leukemia
Fig 1. Structures of scutellarin and scutellarein.

(
APL) cells, Chen group showed that adenanthin directly targets
biotinylated analog (P6) as blank probe (Fig. 2 and Fig. 3). To
make the titled probes, we started from compound , which was
obtained by protecting 5,6-dihydroxyl group of scutellarein (
with diphenyl methyl group. Probe P4 was achieved by the
esterification of compound with Biotin followed with
deprotection of diphenyl methyl group. Linker was condensed
with Biotin to give blank probe P6. Intermediate was
achieved by the etherification of methyl chloroacetate with
compound followed with hydrolysis of methyl ester. Finally,
probe P10 was obtained by conjugation of intermediate with
the conserved cysteines of Prx I and Prx II and thus inhibits
their peroxidase activities in contributing APL differentiation
2
1
)
[
9].
2
5
8
2
8
P6 followed with deprotection of diphenyl methyl group (See
supporting information for the details).
With these probes in hand, we first investigated their stability
both in cells and culture medium. In 6 hours, probes P4 and
P10 were quite stable while half degradation of them occurred
upon 24 hours (Fig. S1). Next, we found that probes P4 and
P10 display the antiproliferative activities of Hela cells
comparable to scutellarein in a concentration range of 50-400
µ
M (Fig. S2). Furthermore, P10 can act as scutellarein in the
Fig 2. Structures of probes for discovering scutellarein
arrest in G2/M and apoptosis in Hela cells (Fig. S3 and Fig. S4).
These data indicated that these probes were suitable for finding
out the molecular target of scutellarein.
interacting proteins.
Using a strategy of chemical biology, we herein report the
design and synthesis of biotinylated scutellareins as probes (Fig.
To discover the direct targets of scutellarein, we performed a
pull-down assay (biotin-streptavidin system) to extract interest
proteins from total lysate of Hela cells, which were incubated in
2
), which can be applied to discover scutellarein interacting
proteins. We also demonstrate that scutellarein can interact with
tumor-specific pyruvate kinase-M2 (PKM2) and thus inhibit its
activity. In terms of their mechanism of action, we conclude
that scutellarein (or scutellarin) may serve as PKM2 inhibitors,
which can directly inhibit PKM2 activity in the cytoplasm; on
the other hand, it may also promote PKM2 translocating into
nucleus where it participates in transcriptional regulation
resulting in the arresting in G2/M and the following apoptosis
of Hela cells.
advance for 6 hours with probes P4, P6 and P10, respectively.
The proteins bound to these probes were extracted from
streptavidin beads and then subjected to SDS-PAGE for
visualization with silver staining. As shown in Fig. 4, some
bands from the extracts of both probes P4 and P10 appear more
abundant than those from the extracts of blank probe P6
.
Especially, one of the bands from probe P10 (marked with a red
asterisk in Fig. 4A) shows unique and more distinguished by
comparison with the other two lanes. This band was sliced and
then in-gel trypsinized for subsequent mass spectroscopic
analysis. Finally, it was identified to be pyruvate kinase M2
(PKM2) (Fig. 4B).
S
H
OH
OH
O
NH
HO
HO
O
O
O
O
O
O
O
O
O
O
H
HN
Ph2CCl2
Biotin
O
DCC, DMAP
OH
O
OH
OH
3
1
, Scutellarein
2
S
H
O
NH
HO
O
O
O
H
HN
TFA
O
DCM
HO
OH
P4
O
NH2
H2N
O
S
H
O
H
N
5
O
NH
Biotin
+
Cl
O
H2N
O
Et3N
O
H
HN
O
P6
O
O
OH
O
O
O
OH
O
O
O
O
O
O
O
O
O
O
LiOH
ClCH2COOCH3
K2CO3
MeOH/H2O
O
O
OH
OH
OH
8
2
7
O
S
H
H
N
O
O
O
NH
N
H
O
Cl
O
O
O
O
O
H
HN
O
Fig 4. Identification of PKM2 as a potential target of
scutellarein. (A) Silver staining of SDS-PAGE of cellular
proteins from extracts of probes P4, P10 and P6; (B) Mass
P6, NMM
9
OH
O
O
S
H
spectroscopic analysis of a protein with a molecular weight
above 48 kDa (indicated by a red asterisk), which was identified
as PKM2.
H
N
O
O
NH
N
H
O
TFA
HO
HO
O
O
H
HN
O
DCM
P10
OH
O
To validate whether PKM2 is the direct target of scutellarein,
we performed a co-immunoprecipitation assay using probe P10
and streptavidin beads. We treated Hela, MCF-7 or A549 cells
Fig 3. Synthetic route for biotinylated probes.
To identify potential targets of scutellarein, we prepared two
with 100
Hela was no P10 added as a negative control. After the lysis of
cells, we performed immunoprecipitation using streptavidin
µM of P10 for 6 hours, respectively. One cell line of
biotinylated scutellareins (P4 and P10) as probes and one
2

beads and then western-blot analysis against PKM2. The results
demonstrate that P10 can pull down PKM2 in different cells
Fig. 5A). To further confirm this interaction, we treated Hela
cells with or without biotin in presence of 100 P10 for 6
(
µ
M
hours. Co-immunoprecipitation assay was performed and show
that the interaction between P10 and PKM2 is indeed specific.
Therefore, we conclude that PKM2 is a direct target of
scutellarein.
Fig 6. Scutellarin and scutellarein inhibit the metabolic rate of
glucose and lactate. (A) Glucose consumption and lactate
production in Hela cells treated with scutellarin and scutellarein.
(
B) Glut1 expression in Hela cells treated with different
concentration of sctuellarein.
Because of the key role of PKM2 in Warburg effect, we
reasoned that scutellarein can decrease the glycolytic flux by
the inhibition of PKM2. Cellular glucose consumption/lactate
production was determined when Hela cells were incubated
with scutellarin/scutellarein or shikonin for 6 hours. The results
showed that scutellarin/scutellarein inhibited the cellular
Fig 5. Verification of PKM2 as a direct target of scutellarein by
co-immunoprecipitation assay using probe P10 and streptavidin
beads. (A) Probe P10 interacts with PKM2 in Hela, MCF-7 and
A549 cells; (B) Competitive assay show that the interaction of
P10 and PKM2 became dose-dependently weak when adding
Biotin in Hela cells.
glycolytic rate in
a
concentration-dependent manner (a
M). We also found that
Pyruvate kinase M2 (PKM2) is a key regulator of Warburg
effect (aerobic glycolysis) in cancer cells, which catalyzes the
last step of glycolysis [10, 11]. Although the mammalian
pyruvate kinase (PK) family include four isoforms (PKL, PKR,
PKM1 and PKM2), PKM2 is the dominant isoform expressed
in cancer or other proliferating cells [12]. Furthermore, PKM2
display the crucial role in glycolytic and non-glycolytic
functions for providing cancer cells with growth and survival
advantage [10]. Therefore, we next to investigate how
scutellarein can inhibit cancer cells growth through PKM2.
Recombinant PKM2 and PKM1 were expressed and purified,
respectively. An enzymatic assay was performed to evaluate the
role of scutellarein for PKM2 according to the reported method
concentration range of 100-400
µ
scutellarein did not decrease the expression level of glucose
transporter 1 (Glut1), which further confirmed that scutellarein
decreased the metabolic rate of glucose and lactate by inhibiting
PKM2 in Hela cells [14].
Besides the well-known cytosolic function of PKM2, its
nuclear function that PKM2 can translocate to the nucleus to
function as a transcriptional coactivator, has been recently
investigated [15, 16]. To find out whether scutellarein can affect
the translocation of PKM2 to the nucleus, we checked the
PKM2 level in nuclear or cytosolic cell fraction in presence of
scutellarein. As shown in Fig. 7A, scutellarein show a time-
dependent manner (from 2 to 8 hours) to promote the nuclear
translocation of PKM2 when Hela cells were treated with 100
[
13, 14]. As shown in Table 1, scutellarein displayed a similar
PKM2 inhibition (12.5 µM) and selectivity (8 folds) compared
µ
M scutellarein in different time duration. Additionally,
scutellarein also show a dose-dependent manner (from 0 to 400
M) in this increasing nuclear translocation of PKM2 when
to a reported synthetic inhibitor, compound 3 [13]. However, a
natural product called shikonin [14] was shown better PKM2
inhibition and selectivity than that of scutellarein. As the
enzymatic assay of PKM2 was used a lactate dehydrogenase
µ
Hela cells were treated with different concentrations of
scutellarein for 6 hours (Fig. 7B).
(
LDH) coupled assay, an additional LDH assay must be carried
out for ruling out that LDH activity is not affected by
scutellarein [13, 14]. The results showed that LDH activity was
not inhibited by scutellarein (Fig. S5).
Table 1 IC50 values and selectivity of PKM2 inhibitors.
Fig 7. Scutellarein promote the translocation of PKM2 to the
nucleus. (A) Hela cells were treated with 100 µM scutellarein in
different time duration. (B) Hela cells were treated with
different concentration of scutellarein for 6 hours.
3

the Engineering Center of Natural Science Foundation of
Department of Education of Guizhou Province (Microbiology
&
Biochemical Pharmacy, QJHKYZ[2015]338), and Training
Program of Innovation and Entrepreneurship for
Undergraduates (No. 201610660010).
Supplementary Material
Fig 8. Scutellarein promote the nuclear translocation of PKM2
Supplementary data associated with this article can be found,
in the online version, at http://
though the MEK/ERK/PIN1 pathway.
To further investigate this mechanism, we checked the effect
of scutellarein on MEK/ERK/PIN1 pathway, which is the well-
established pathway for nuclear translocation of PKM2 [16].
Scutellarein can increase the expression of mitogen-activated
protein kinase kinase (MEK) when Hela cells were treated with
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, Bax/Bcl-2 pathway.
In summary, we have demonstrated that biotinylated
[
[
[
[
[
[
[
scutellarein P10 is a practical and useful tool to study
scutellarin/scutellarein interacting proteins in treating cancer.
We further show that PKM2 is one of the direct targets of
scutellarein and scutellarein is a selective inhibitor of PKM2,
which can decrease glycolytic metabolism in Hela cells by
inhibiting cytosolic PKM2; on the other hand, scutellarein may
also participate in regulating cell cycle and apoptotic proteins
by activating MEK/ERK/PIN1 signaling pathway to promote
the nuclear translocation of PKM2. This finding will facilitate
the research and development of more potent anticancer agents
based on the scaffold of scutellarin.
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We are grateful for financial support from National Natural
Science Foundation of China (No. 21302027 and 21662010),
One Thousand Talents Program of Guizhou Province, the
Innovation Team of Natural Science Foundation of Department
of Education of Guizhou Province (No. QJHRCTDZ[2015]57),
4

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Scutellarin inhibits Hela cell growth and
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pyruvate kinase M2
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Lin You, Hong Zhu, Chun Wang, Fang Wang, Yongjun Li, Yan Li, Yonglin Wang, Bin He
5