The oxidation of (-)-epigallocatechin-3-gallate inhibits T-cell acute lymphoblastic leukemia cell line HPB-ALL: Via the regulation of Notch1 expression

Article abstract of DOI:10.1039/c9ra08459b

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and commonly associated with activating mutations in the Notch1 pathway. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant and active catechin and has been shown to regulate Notch signaling. Taking into account the highly oxidizable and unstable of EGCG, we proposed that EGCG oxides may have greater potential to regulate Notch signaling than EGCG. In this study, we isolated and identified EGCG oxides (compound 2-4), using a chemical oxidation strategy, and evaluated for cytotoxicity against T-cell acute lymphoblastic leukemia cell line (HPB-ALL) by using the MTS assay. We found compound 3 significantly induced cell proliferation inhibition (38.3858 ± 1.67106 μM), cell apoptosis and cell cycle arrest in a dose-dependent manner. Remarkably, compound 3 inhibited expression of Notch1 compared with EGCG in HPB-ALL cells. Meanwhile, we found that compound 3 significantly inhibited c-Myc and Hes1, which are downstream target genes of Notch1. The findings demonstrate for the first time that an oxidation product of EGCG (compound 3) inhibits T-cell acute lymphoblastic leukemia cell line (HPB-ALL) and is a promising agent for cancer therapy deserving further research.

Full text of DOI:10.1039/c9ra08459b

RSC Advances
View Article Online
View Journal | View Issue
PAPER
The oxidation of (ꢀ)-epigallocatechin-3-gallate
inhibits T-cell acute lymphoblastic leukemia cell
line HPB-ALL via the regulation of Notch1
expression
Cite this: RSC Adv., 2020, 10, 1679
Yu-Na Wang,†a Jing Wang,†a Hao-Nan Yang,a Bang-Lei Zhang,a Pan Zhang,a
a
a
a
ac
abc
*
*
Pei-Yuan Sun, Nin Zhang, Ya Wang, Jun Sheng,
Xuan-Jun Wang
ab
*
and Cheng-Ting Zi
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and commonly
associated with activating mutations in the Notch1 pathway. (ꢀ)-Epigallocatechin-3-gallate (EGCG) is the
most abundant and active catechin and has been shown to regulate Notch signaling. Taking into
account the highly oxidizable and unstable of EGCG, we proposed that EGCG oxides may have greater
potential to regulate Notch signaling than EGCG. In this study, we isolated and identified EGCG oxides
(compound 2–4), using a chemical oxidation strategy, and evaluated for cytotoxicity against T-cell acute
lymphoblastic leukemia cell line (HPB-ALL) by using the MTS assay. We found compound 3 significantly
induced cell proliferation inhibition (38.3858 ꢁ 1.67106 mM), cell apoptosis and cell cycle arrest in
a dose-dependent manner. Remarkably, compound 3 inhibited expression of Notch1 compared with
EGCG in HPB-ALL cells. Meanwhile, we found that compound 3 significantly inhibited c-Myc and Hes1,
which are downstream target genes of Notch1. The findings demonstrate for the first time that an
oxidation product of EGCG (compound 3) inhibits T-cell acute lymphoblastic leukemia cell line (HPB-
ALL) and is a promising agent for cancer therapy deserving further research.
Received 16th October 2019
Accepted 23rd December 2019
DOI: 10.1039/c9ra08459b
rsc.li/rsc-advances
apoptosis and cellular metabolism by driving the expression of
c-Myc, Hes1 and regulating other critical oncogenic signaling
pathways.9–12 Many preclinical and clinical studies have
explored the efficacy of targeting Notch in T-ALL, most
commonly with g-secretase inhibitors (GSIs).13
1. Introduction
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive
blood malignancy, which is usually reected as an invasive
tumor and accounts for approximately 25% and 15% of adult
acute lymphoblastic leukemia in adult and pediatric cohorts,
respectively.1,2 Currently, T-ALL treatment schedules consist of
high-dose chemotherapy, which is oen associated with lack
selectivity and displays signicant toxic side effects.3–5 There is
an urgent clinical need for optimized treatment stratication
and more effective antileukemic drugs for the treatment of
human T-ALL. Development of targeted drugs, increase of
tumor selectivity and decrease of toxicity are the key topics
which are currently being studied.
Natural products with various structures, unique biological
activities and specic selectivity have served as important
sources of lead compounds for antitumor agents which have
been developed for clinical use.14 (ꢀ)-Epigallocatechin-3-gallate
(EGCG, 1, Fig. 1) is the most abundant catechin (accounting for
approximately 50% of total catechins) and has been reported to
have stronger physiological activities.15,16 Previously, some
studies have reported that several proteins have been identied
as EGCG direct interactors including the trans-membrane
receptor 67LR, metalloproteinases (MMPs), Pin1 and TGFR-
II13.17 In addition, EGCG has the activity of regulating Notch
signaling in the development of tumor angiogenesis and colo-
rectal cancer.18–21 However, the use of EGCG is oen hindered by
problems such as easy oxidation, ready degradation in aqueous
solutions and the poor intestinal absorbance.16,22
Statistically, activating mutations in Notch1 occur in over
50% of T-ALLs,6–8 The deregulated Notch signaling affects
diverse cellular processes involved in growth, proliferation,
aKey Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural
University, Kunming, 650201, China. E-mail: shengj@ynau.edu.cn; wangxuanjun@
gmail.com; zichengting@126.com
bCollege of Science, Yunnan Agricultural University, Kunming, 650201, China
cState Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan,
Yunnan Agricultural University, Kunming, 650201, China
There have been many studies reporting that EGCG encap-
sulated into nanocarriers leads to its strengthened stability and
higher therapeutic effects.23,24 In this study, we isolated and
identied EGCG oxides by a chemical strategy, tested the ability
† These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 1679–1684 | 1679

View Article Online
RSC Advances
Paper
of oxides with EGCG to inhibit T-cell acute lymphoblastic
leukemia cell line (HPB-ALL), and further developed more
effective antileukemic drugs for the treatment of human T-ALL.
2. Results and discussion
2.1. Chemical oxidation and structural determination
The chemical oxidation of EGCG has been reported under
various oxidative conditions, including K3[Fe(CN)6],25 CuSO4,26
and CuCl2.25 In this research, the oxidation of EGCG was based
on X. C. Wan and R. G. Bailey et al. method27,28 with some
modication. EGCG was reacted with K3[Fe(CN)6]/NaHCO3 at
25 ꢂC for 15 min to yield the EGCG oxides 2–4 in 1.3–6.3% yields
(Fig. 1).
Compound 2 was obtained as a white amorphous powder
and shown to have the same molecular formula as EGCG by its
1
HRESIMS. In the H-NMR spectra (Table 1), the proton at C-2
(5.52 ppm, d, J ¼ 2.4 Hz) and C-3 (4.96 ppm, d, J ¼ 2.0 Hz)
appear as a double peak, indicating a cis-relationship between
H-2 and H-3. Compound 3 was isolated as a yellow amorphous
powder. The negative HRESIMS at m/z 913.7339 [M–H]ꢀ (calcd
Fig. 1 The chemical structures of EGCG (1) and its oxides (2–4).
Table 1 1H-NMR and 13C-NMR data for EGCG and EGCG oxides 2–4
EGCG (d)
Compound 2 (d)
Compound 3 (d)
Compound 4 (d)
C position
dC
dH
dC
dH
dC
dH
dC
dH
2
3
4
78.6
69.9
26.8
5.56 (brs)
4.92 (s)
3.03–2.92 (m, H)
78.6
69.9
26.8
5.52 (d, J ¼ 2.4 Hz)
4.96 (d, J ¼ 2.0 Hz)
3.02–2.90 (m, H)
76.3
69.0
26.9
5.23 (brs)
4.90 (s)
4.72 (s)
94.0
69.3
27.1
4.47 (s)
4.47 (s)
3.16–3.12 (m, H)
5 (7)
6
8
157.8
96.5
95.9
157.9
96.4
95.8
157.7
96.6
95.9
157.7
96.8
95.8
5.94 (s)
5.94 (s)
5.93 (s)
5.93 (s)
5.88 (s)
5.87 (s)
5.92 (d, J ¼ 2.3 Hz)
5.90 (d, J ¼ 2.3 Hz)
9
157.8
99.4
157.9
99.4
157.7
99.0
157.0
99.7
156.5
54.9
10
10
130.8
106.9
146.7
133.8
146.7
106.9
130.7
106.8
146.7
133.8
146.7
106.8
129.5
108.3
146.9
134.4
146.9
108.3
129.5
76.3
20
6.49 (s)
6.49 (s)
6.48 (s)
6.48 (s)
6.74 (s)
6.74 (s)
3.34 (s)
30
110.2
148.0
200.3
124.0
128.0
109.2
140.1
130.9
148.5
114.9
40
500
600
5.96 (s)
6.42 (s)
100 (1000)
200
300
69.0
20.8
5.23–5.25 (m)
3.31–3.29 (m)
4
500
157.3
96.6
157.7
112.3
99.0
108.3
145.4
134.4
145.4
108.3
600
5.91 (d, J ¼ 2.3 Hz)
700 (900)
800 (8000)
1000000 (10000)
2000
95.7
99.2
78.5
66.4
27.0
157.7
96.6
157.0
167.7
121.1
110.2
146.3
139.8
5.75 (d, J ¼ 2.3 Hz)
6.74 (s)
5.45 (brs)
4.57 (s)
2.89–2.82 (m)
3000
4
5000 (7000)
6000
6.74 (s)
5.80 (d, J ¼ 2.3 Hz)
9000
Galloyl 0
Galloyl 1
Galloyl 2 (6)
Galloyl 3 (5)
Galloyl 4
167.7
121.4
110.2
146.7
139.8
167.6
121.4
110.2
146.7
139.7
167.8
121.4
110.3
146.3
139.8
6.94 (s, 2H)
6.93 (2H, s)
6.91 (s, 2H)
6.95 (s, 2H)
1680 | RSC Adv., 2020, 10, 1679–1684
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
for C44H34O22, 913.7340) supporting a molecular formula of Notch1 signaling pathway. To investigate the effects of
1
C
44H34O22. The H-NMR and 13C-NMR spectra of compound 3 compound 3 on Notch1 signaling pathway, we examined the
(Table 1) corresponded to the data reported in the literature.29 expression of Notch1 and the downstream proteins in HPB-ALL
Compounds 4, a yellow amorphous powder, was assigned the cells (Fig. 3). As shown in Fig. 3B–F, HPB-ALL cells were treated
molecular formula of C44H34O23 determined by the negative with compound 3 at the concentration of 5, 10, 20, 40, 60 mM for
HRESIMS at m/z 929.1450 [M–H]ꢀ (calcd for C44H34O23, 12 h and the expression level of Notch1, cleaved-Notch1, c-Myc
929.1490), as well as from its NMR data (Table 1), which dis- and Hes1 were signicantly decreased.7 Meanwhile, we also
played a signal pattern similar to the data reported in the found that the expression of proliferation marker Ki67 was
literatures.30,31
signicantly reduced (Fig. 3B and G). These ndings suggest that
compound 3 inhibited the proliferation of HPB-ALL cells may be
through down-regulating the expression of Notch1 and Ki67.
2.2. Compound 3 is identied as the most potent cytotoxic
compound against HPB-ALL cells
2.4. Compound 3 induces cell cycle G2 arrest in HPB-ALL
cells
We tested the stability of compounds with H2O2 quantitative
assay kit. As shown in Fig. 2A, EGCG was oxidized signicantly.
However, the stability of the compound 2, 3 and 4 was signi-
cantly improved compared with EGCG.
Previous studies have identied several T-ALL cell lines that
are sensitive to GSI treatment.6,32 To identify anticancer activity
of EGCG oxides, we treated HPB-ALL cells with EGCG and its
oxides in different concentrations and measured the cell
viability using MTS assay (Fig. 2B). As shown in Fig. 2B,
compound 3 appeared to be the most potent of these oxides.
The IC50 value of compound 3 was 38.3858 ꢁ 1.67106 mM in the
HPB-ALL cells.
Given that compound 3 inhibited HPB-ALL cells proliferation, we
wondered whether 3 causes cell cycle arrest. We treated the HPB-
ALL cells with compound 3 (5, 10, 20, 40, 60 mM) for 48 h and
analyzed their cell cycle proles by propidium iodide (PI) staining
(Fig. 4). As shown in Fig. 4A and B, in the HPB-ALL cells,
compound 3 signicantly increased the percentage of cells in G2
phase compared to control. Meanwhile, we examined the protein
levels of several cell cycle (CDK2, cyclin E, cyclin D1). As shown in
Fig. 4C–F, compound 3 reduced the expression levels of CDK2
and cyclin E, increased the expression levels of cyclin D1.
2.3. Compound 3 blocks Notch1 processing in HPB-ALL cells
2.5. Compound 3 induces apoptosis in HPB-ALL cells
Cell growth and proliferation can be regulated by Notch1 in T-
ALL.33 Previous studies have shown that EGCG regulates the
inammatory response by targeting Notch.34,35 In addition,
compound 3 is a dimeric EGCG derivative, share a similar
chemical structure with EGCG. Therefore, we speculated that the
cytotoxicity of compound 3 may be partly due to the inhibition of
Previous studies have shown that EGCG exhibits the potential to
promote apoptosis of tumor cells.36,37 Compound 3 has good
Fig. 3 The effect of compound 3 on Notch1 processing and down-
stream signaling pathway in HPB-ALL cells. (A) HPB-ALL cells were
Fig. 2 The stability of compounds and the effects on the proliferation treated with DAPT (1, 5, 10, 20 mM), and EGCG (50, 100, 200 mM) for
of HPB-ALL cells. (A) The HPB-ALL cells were treated with EGCG and 12 h. (B) HPB-ALL cells were treated with compound 3 (5, 10, 20, 40,
compounds 2–4 (3.125, 6.25, 12.5, 25, 50 and 100 mM) for 48 h. Cell 60 mM) for 12 h. The expression levels by western blot (WB) to detect
proliferation was measured by MTS assay. (B) The stability of Notch1, cleaved-Notch1, c-Myc, Hes1 and Ki67. b-Actin was used as
compounds was assessed by determining the H2O2 content using the loading control. (C–G) Quantification of relative cleaved-Notch1,
a H2O2 quantitative assay kit. Data represent the average of three Notch1, c-Myc and Hes1 protein levels. Data represent the average of
independent experiments (mean ꢁ SEM). ***p < 0.001 vs. the control. three independent experiments (mean ꢁ SEM). *p < 0.01, **p < 0.005,
###p < 0.001 vs. EGCG.
***p < 0.001 vs. the control.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 1679–1684 | 1681

View Article Online
RSC Advances
Paper
Fig. 4 Compound 3 induces cell cycle G2 arrest in HPB-ALL cells. (A)
HPB-ALL cells were treated with DAPT (5 mM), EGCG (100 mM) and
compound 3 (5, 10, 20, 40, 60 mM) for 48 h. The cell cycle distribution
was analyzed by FlowJo software (version 7.6). (B) Quantification of
the cell populations in each phase of the cell cycle was presented in
HPB-ALL cells. (C) HPB-ALL cells were treated with compound 3 (5, 10,
20, 40, 60 mM) for 12 h. The protein levels of cell cycle-related protein
were checked by western blot (WB). b-Actin was used as the loading
control. (D–F) Quantification of relative CDK2, cyclin E and cyclin D1
protein levels. Data represent the average of three independent
experiments (mean ꢁ SEM). ***p < 0.001 vs. the control.
Fig. 5 Compound 3 induces apoptosis in HPB-ALL cells. (A) Flow
cytometry of T-ALL cells HPB-ALL after treatment with DAPT (5 mM),
EGCG (100 mM) and compound 3 (5, 10, 20, 40, 60 mM) for 48 h. (B) The
ratio of apoptotic cells in each group are expressed as percentages. (C)
The cells were treated with compound 3 (5, 10, 20, 40, 60 mM) for 12 h.
The expression level of cleaved-caspase 3, caspase 3, cleaved-caspase
9, caspase 9, cleaved-PARP 1, and PARP 1 were detected by western
blot (WB). b-Actin was used as the loading control. (D–F) Quantifica-
tion of relative cleaved-caspase 3, cleaved-caspase 9 and cleaved-
PARP 1 protein levels. Data represent the average of three independent
experiments (mean ꢁ SEM). *p < 0.01, **p < 0.005, ***p < 0.001 vs. the
control.
cytotoxicity against HPB-ALL cell lines, suggesting that
compound 3 mainly induces cell death in addition to causing
cell cycle arrest. To test whether compound 3 induces apoptosis
in HPB-ALL cells, we treated the cells with DAPT (5 mM), EGCG
(100 mM) and compound 3 (5, 10, 20, 40, 60 mM) for 48 h, and
measured the apoptosis of the HPB-ALL cells by FITC Annexin V
staining and ow cytometry. Compound 3 signicantly induced
Annexin V positive apoptotic cells in HPB-ALL cells (Fig. 5A and
B). Moreover, compound 3 induced the expression levels of
caspase 3, caspase 9 and PARP 1, and increased the expression
levels of cleaved-caspase 3, cleaved-caspase 9 and cleaved-PARP
1 (Fig. 5C–F).
cleaved-caspase 9, caspase 9, CDK2 and b-actin were obtained
from Cell Signaling Technology (Beverly, MA, USA). Antibodies
against c-Myc, cleaved-PARP1, PARP1 and Ki67 were obtained
from Abcam (Lake Placid, NY, USA). Antibodies against cyclin
E and cyclin D1 were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). All reagents were commercially avail-
able and used without further purication unless indicated
otherwise. The melting points were measured by an X-4
melting point apparatus and were uncorrected. Mass spectra
(MS) and high-resolution mass spectra (HRMS) data were
recorded on an Agilent 6540 Q-Tof (ESIMS) (Agilent, Cal-
ifornia, USA). Proton nuclear magnetic resonance (1H-NMR)
3. Experimental
3.1. Materials and methods
and carbon-13 nuclear magnetic resonance
(
13C-NMR)
spectra were recorded on Bruker AVANCE III 500 MHz
(Bruker BioSpin GmbH, Rheinstetten, Germany) instruments,
using tetramethylsilane (TMS) as an internal standard:
chemical shis (d) are given in ppm, coupling constants (J)
in Hz, the solvent signals were used as references (CD3OD: dC
¼ 49.0 ppm; residual CH3OH in CD3OD: dH ¼ 4.78 ppm). Silica
gel (200–300 mesh) for column chromatography and silica
GF254 for thin-layer chromatography (TLC) were produced by
Qingdao Marine Chemical Company (China).
(ꢀ)-Epigallocatechin-3-gallate was purchased from Chengdu
Proifa Technology Development Co., Ltd (Chengdu, China); 3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt (MTS) purchased from
Promega (Madison, WI, USA). H2O2 quantitative assay kit was
provided by sangon biotech (Shanghai, China). HPB-ALL cell
line were purchased from the American Type Culture Collec-
tion (ATCC, Manassas, Virginia, USA); antibodies against
cleaved-Notch1, Notch1, Hes1, cleaved-caspase 3, caspase 3,
1682 | RSC Adv., 2020, 10, 1679–1684
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
amounts were loaded 8% SDS-polyacrylamide gel and then
transferred onto polyvinylidene uoride (PVDF) membranes
(EMD Millipore Corporation, Merck Life Sciences, KGaA,
Darmstadt, Germany). Membranes containing protein blots
were blocked with 5% non-fat milk in TBST at room tempera-
ture for 1 h and then incubated with the primary antibodies at
3.2. Oxidation of EGCG and characteristic data of
compounds 2–4
To a solution of EGCG (200 mg, 0.45 mmol) in 10 mL of distilled
water was added dropwise a solution of potassium hex-
acyanoferrate (III) (300 mg, 0.90 mmol) and solium hydrogen
carbonate (100 mg, 1.2 mmol) in 10 mL of distilled water at
25 ꢂC, pH 7.0 for 15 min. Then, the pH of resulting mixture was
adjusted to 2.0 by addition of saturated aqueous citric acid (2
mL). The reaction mixture was extracted with ethyl acetate
(10 mL ꢃ 3), and the combined organic layer was dried over
sodium sulfate (s). The solvent was evaporated under vacuum
and the residue was puried by column chromatography (silica
gel, chloroform : methanol ¼ 4 : 1 / 1 : 1, in 0.2% acetic acid)
to afford compound 2 (4.9 mg, 2.5%), compound 3 (12.6 mg,
6.3%), and compound 4 (2.5 mg, 1.3%).
ꢂ
4 C overnight. Following a wash with TBST, membranes were
incubated with secondary antibodies for 1 h. Detection was
performed using a FluorChem E System (ProteinSimple, Santa
Clara, CA, United States).
3.6. Cell cycle analysis
HPB-ALL cells were seeded in the 6-well plates at a density of 1.0
ꢃ 106 cells per well, harvested, and washed twice with PBS. Aer
The cells were synchronized for 48 h in medium containing 3%
FBS in the presence or absence of DAPT, EGCG and compound
3,ꢂand then washed in ice-cold PBS and xed in 70% ethanol at
4 C for 24 h. Then the cells were washed twice with cold PBS
and stained in buffer containing PI (10 mg mLꢀ1) and RNase.
Aer 30 min incubation in the dark on ice, cell cycle distribu-
tions were detected and analyzed by ow cytometry (BD FACS-
Calibur, USA).
1
Compound 2: white amorphous powder, H-NMR (CD3OD,
500 MHz) and 13C-NMR (CD3OD, 125 MHz) data: see Table 1;
ESIMS: m/z 457 [M–H]ꢀ, HRESIMS was calculated for C22H17O11
[M–H]ꢀ 457.0849 and was found to be 457.0850. Compound 3:
yellow amorphous powder, 1H-NMR (CD3OD, 400 MHz) and
13C-NMR (CD3OD, 100 MHz) data: see Table 1; ESIMS: m/z 457
[M–H]ꢀ, HRESIMS was calculated for C44H33O22 [M–H]ꢀ
913.7340 and was found to be 913.7339. Compound 4: yellow
amorphous powder, 1H-NMR (CD3OD, 500 MHz) and 13C-NMR
(CD3OD, 125 MHz) data: see Table 1; ESIMS: m/z 457 [M–H]ꢀ,
HRESIMS was calculated for C44H33O23 [M–H]ꢀ 929.1490 and
was found to be 929.1450.
3.7. Cell apoptosis assay
HPB-ALL cells were seeded in the 6-well plates at a density of
1.0 ꢃ 106 cells per well. Aer the cells were pretreated with
DAPT, EGCG and compound 3 and incubated for 48 h in
medium containing 3% FBS. Following collection and suspen-
3.3. Measurement of the stability of compounds
The medium containing the different compounds (50 mM) was sion in binding buffer, the cells were incubated with 5 mL of
incubated at 37 ꢂC for 30 min. According to the instructions, the Annexin V and 5 mL of PI (BD Biosciences) for 15 minutes in
medium and operating uid was transferred into a 96-well darkness. Detection was performed using a ow cytometry (BD
plate. The plate was shaken for 30 s and le at room tempera- FACSJazz TM Cell Sorter, 655487, USA).
ture for 20 min. The optical density (OD) values were measured
at 595 nm with a Flex Station 3 Multi-Mode Microplate Reader
(Molecular Devices).
4. Conclusions
In summary, we isolated and identied EGCG oxides
(compound 2–4), using a chemical oxidation strategy, and
3.4. Cell toxicity assay
HPB-All cells were placed into 96-well plates at a density of evaluated for cytotoxicity against T-cell acute lymphoblastic
2.0 ꢃ 105 cells per well and treated with medium free of leukemia cell line (HPB-ALL) by using MTS assay. Among
phenol red and different concentrations of EGCG and EGCG them, compound 3 showed the highest anticancer activity with
oxides. Cells was incubated at 37 ꢂC for 48 h. 20 mL of MTS its IC50 values at 38.3858 ꢁ 1.67106 mM. We founding that
solution was transferred to each well, and then the plate was compound 3 inhibited the proliferation of HPB-ALL cells may
ꢂ
incubated at 37 C for 4 h. The OD values were measured at be through down-regulating the expression of Notch1 and
490 nm with Flex Station 3 Multi-Mode Microplate Reader Ki67. Compound 3 induced cell cycle G2 arrest in HPB-ALL
(Molecular Devices).
cells, and also induced the expression levels of the apoptotic
maker protein (caspase 3, caspase 9 and PARP 1). We
demonstrated for the rst time that an oxidation product of
EGCG (compound 3) inhibits T-cell acute lymphoblastic
leukemia cell line HPB-ALL via the regulation of Notch1
expression.
3.5. Western blot analysis
HPB-ALL cells were seeded in the 100 mm plates at a density of
6.0 ꢃ 106 cells per well and treated with compound 3 at
different concentrations in cell culture asks, and then the cells
were harvested and lysed in lysis buffer to assess the expression
of Notch1, c-Myc, Hes1, caspase 3, caspase 9, PARP 1, Ki67,
cyclin E, cyclin D1 and CDK2. Extracted proteins samples were
Conflicts of interest
quantied using bicinchoninic acid (BCA) assays; equal The authors declare no conict of interest.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 1679–1684 | 1683

View Article Online
RSC Advances
Paper
16 X. Zhang, J. Wang, J. M. Hu, Y. W. Huang, X. Y. Wu, C. T. Zi,
X. J. Wang and J. Sheng, Molecules, 2016, 21, 620.
17 A. Negri, V. Naponelli, F. Rizzi and S. Bettuzzi, Nutrients,
2018, 10, 1–24.
18 L. T. Gu, J. Yang, S. Z. Su, W. W. Liu, Z. G. Shi and
Q. R. Wang, Neurochem. Res., 2015, 40, 1211–1219.
19 H. Xie, J. Q. Sun, Y. Q. Chen, M. Zong, S. J. Li and Y. Wang,
Oxid. Med. Cell. Longevity, 2015, 2015, 1–10.
Acknowledgements
We are grateful to the National Nature Science Foundation of
China for nancial support (No. 21602196); the Yunnan
Provincial Science and Technology Department (No. 2017ZF003,
2017FD084, and 2017FG001-046).
20 Y. Gao, G. O. Rankin, Y. Tu and Y. C. Chen, Int. J. Oncol.,
2016, 48, 281–292.
Notes and references
1 A. A. Ferrando, D. S. Neuberg, J. Staunton, M. L. Loh,
C. Huard and S. C. Raimondi, Cancer Cell, 2002, 1, 75–87.
2 E. Baghbani, B. Baradaran, F. Pak, L. Mohammadnejad,
D. Shanehbandi, B. Mansoori, V. Khaze, N. Montazami,
A. Montazami and P. Kokhaei, Biomed. Pharmacother.,
2017, 86, 41–47.
21 H. Jin, W. Gong, C. Zhang and S. Wang, OncoTargets Ther.,
2013, 6, 145–153.
22 S. Kitao, T. Matsudo, M. Sasaki, T. Horiuchi and H. Sekine,
Biosci. Biotechnol. Biochem., 1995, 59, 2167–2169.
23 O. Krupkova, S. J. Ferguson and K. Wuertz-Kozak, J. Nutr.
Biochem., 2016, 37, 1–12.
3 A. J. Barrett, M. M. Horowitz, B. H. Pollock, M. J. Zhang,
M. M. Bortin and G. R. Buchanan, N. Engl. J. Med., 1994,
331, 1253–1258.
4 J. M. Goldberg, L. B. Silverman, D. E. Levy, V. K. Dalton,
R. D. Gelber and L. Lehmann, J. Clin. Oncol., 2003, 21,
3616–3622.
24 G. Andreia, I. Frias, N. A. Neves, M. Pinheiro and S. Reis,
BioMed Res. Int., 2017, 2017, 1–15.
25 Y. Takino, H. Imagawa, Y. Aoki and T. ocirc ko Ozawa, Agric.
Biol. Chem., 1963, 27, 562–568.
26 T. Shii, M. Miyamoto, Y. Matsuo, T. Tanaka and I. Kouno,
Chem. Pharm. Bull., 2011, 59, 1183–1185.
5 P. V. Vlierberghe and A. Ferrando, J. Clin. Invest., 2012, 122,
3398–3406.
6 A. P. Weng, A. A. Ferrando, L. Woojoong, J. P. Morris,
L. B. Silverman and S. I. Cheryll, Science, 2004, 306, 269–271.
7 C. Grabher, H. Von Boehmer and A. T. Look, Nat. Rev. Cancer,
2006, 6, 347–359.
27 X. C. Wan, H. E. Nursten, C. Ya, A. L. Davis, J. P. G. Wilkins
and A. P. Davies, J. Sci. Food Agric., 1997, 74, 401–408.
28 R. G. Bailey, H. E. Nursten and I. Mcdowell, J. Sci. Food Agric.,
1993, 63, 455–464.
29 J. P. de Mello, F. Petereit and A. Nahrstedt, Phytochemistry,
1996, 41, 807–813.
8 I. Pinto, M. Duque, J. Gonçalves, P. Akkapeddi, M. L. Oliveira,
R. Cabrita, J. A. Yunes, S. K. Durum, J. T. Barata and
R. Fragoso, Oncogene, 2019, 1–12.
9 V. Giambra, C. R. Jenkins, H. Wang, S. H. Lam,
O. O. Shevchuk and O. Nemirovsky, Nat. Med., 2012, 18,
1693–1698.
30 S. Valcic, A. Muders, N. E. Jacobsen, D. C. Liebler and
B. N. Timmermann, Chem. Res. Toxicol., 1999, 12, 382–386.
31 T. Tanaka, S. Watarumi, Y. Matsuo, M. Kamei and I. Kouno,
Tetrahedron, 2003, 59, 39–47.
32 T. Palomero, M. L. Sulis, M. Cortina, P. J. Real, K. Barnes,
M. Ciofani, E. Caparros, J. Buteau, K. Brown, S. L. Perkins,
G. Bhagat, A. M. Agarwal, G. Basso, M. Castillo, S. Nagase,
´
10 S. Jarriault, B. O. Le, E. Hirsinger, O. Pourquie, F. Logeat and
C. F. Strong, Mol. Cell. Biol., 1998, 18, 7423–7431.
11 S. Weijzen, P. Rizzo, M. Braid, R. Vaishnav, S. M. Jonkheer,
A. Zlobin, B. A. Osborne, S. Gottipati, J. C. Aster,
W. C. Hahn, M. Rudolf, K. Siziopikou, W. M. Kast and
L. Miele, Nat. Med., 2002, 8, 979–986.
12 A. P. Wen, Genes Dev., 2006, 20, 2096–2109.
13 N. Takebe, D. Nguyen and S. X. Yang, Pharmacol. Ther., 2014,
141, 140–149.
14 C. T. Zi, Y. S. Gao, L. Yang, S. Y. Feng, Y. Huang, L. Sun,
Y. Jin, F. Q. Xu, F. W. Dong, Y. Li, Z. T. Ding, J. Zhou,
Z. H. Jiang, S. T. Yuan and J. M. Hu, Front. Chem., 2019, 7,
434.
15 C. Cabrera, R. Gimenez and M. C. Lopez, J. Agric. Food Chem.,
2003, 51, 4427–4435.
˜
´
¨
C. Cordon-Cardo, R. Parsons, J. C. Zuniga-Pucker,
M. Dominguez and A. Ferrando, Nat. Med., 2007, 13, 1203–
1210.
33 M. Paganin and A. Ferrando, Blood Rev., 2011, 25, 83–90.
34 T. F. Wang, Z. M. Xiang, Y. Wang, X. Li, C. Y. Fang, S. Song,
C. L. Li, H. S. Yu, H. Wang, L. Yan, S. M. Hao, X. J. Wang and
J. Sheng, Front. Immunol., 2017, 8, 433.
35 Y. W. Huang, Q. Q. Zhu, X. Y. Yang, H. H. Xu, B. Sun,
X. J. Wang and J. Sheng, FASEB J., 2018, 33, 1–12.
36 W. H. Yang, Y. C. Fong, C. Y. Lee, T. R. Jin, J. T. Tzen, T. M. Li
and C. H. Tang, J. Cell. Biochem., 2011, 112, 1601–1611.
37 Y. Q. Wang, J. L. Lu, Y. R. Liang and Q. S. Li, Molecules, 2018,
23, 23–34.
1684 | RSC Adv., 2020, 10, 1679–1684
This journal is © The Royal Society of Chemistry 2020