supplemental figure 1: ascorbate induces apoptosis and inhibits … · 2018-02-06 · supplemental...
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1
Supplemental Figure 1: Ascorbate induces apoptosis and inhibits
proliferation of gastric cancer cells. (A) Quantification of cell apoptosis
in the indicated cells treated with ascorbate (4mM, 2h) were determined
by Annexin V/propidium iodide (PI) assays. (B) The intracellular ATP
level in the indicated cells treated with ascorbate for 1h was measured. (C)
Immunoblotting of γ-H2AX in MGC803 cells after treatment with
ascorbate for 2h. β-Actin was used as a loading control. (D)
Paraffin-embedded tumor sections were stained with anti-Ki67 and
cleaved caspase 3 antibody (scale bar: 50 μm), the proliferation and
apoptotic index was quantified. Data in A, B and D (n = 4 for A, B and n
= 6 for D) are presented as mean ±S.D. *P < 0.05 versus control.
Supplemental Figure 2: Ascorbate acts as pro-oxidant in gastric
cancer. (A) Quantification of ROS contents in the presence and absence
of ascorbate (1mM or 2mM for 1h) in the indicated cells as detected by
the fluorescent probe DCF-DA. (B) Relative levels of NADPH/NADP+
in the indicated cells in the presence and absence of ascorbate at 1mM for
1h. (C, D) Pretreatment with NAC or catalase attenuated pro-apoptosis
effects of ascorbate in the indicated cells. (E) Apoptosis analysis of
SGC7901 cells treated with DFO (200μM) and DTPA (1mM) for 3h
followed by 2h exposure to ascorbate (4mM) in the continued presence of
these chelators. (F) Apoptosis analysis of SGC7901 cells in the presence
2
or absence of red blood cells (RBC) at 25% hematocrit treated with
ascorbate at 2mM for 2h. Data in A, B, C, D, E and F (n = 3) are
presented as mean ±S.D. *P < 0.05 versus control. NS, non-significant.
Supplemental Figure 3: GLUT1 mediates anti-tumor activity of
ascorbate in gastric cancer cells. (A) Concentrations of glucose and
lactate were determined with the SBA40C Biosensor. The glucose uptake
was determined as the concentration in the fresh medium minus that in
the cell culture medium for 24h, while the lactate production was
calculated as the concentration in the cell culture medium for 24h minus
that in the fresh medium. (B) qPCR analysis of HK2, Aldolase, PFK1,
PKM2 and LDH-A in the indicated cells. (C) Relative levels of DCF-DA,
glutathione and ATP in HGC27 cells in the presence and absence of
ascorbate at 1mM for 1h. (D) Immunoblotting of γ-H2AX in HGC27 cells
treated with ascorbate at 2mM and 4mM for 2h. β-Actin was used as a
loading control. (E-H) qPCR analysis of SVCT1, SVCT2, Glut3 and
Glut4 mRNA level in the indicated cells. (I) Immunoblotting of SVCT1,
SVCT2, GLUT3 and GLUT4 in the indicated cells. β-Actin was used as a
loading control. (J) qPCR analysis of Glut1 mRNA level in the indicated
cells. Data in A, B, C, E, F, G, H and J (n = 3) are presented as mean
±S.D. *P < 0.05 versus control.
3
Supplemental Figure 4: Efficiency of GLUT1 manipulation. (A)
Western blot analysis of knockdown efficiency of GLUT1. The
membranous proteins were isolated. β-Actin and Na+/K+ ATPase was
used as cytoplasmic and membranous loading control, respectively. (B)
Flow cytometry analysis of GLUT1 fluorescence in the indicated cells. (C)
Colony formation of HGC27 cells with enforced GLUT1 expression after
ascorbate treatment. (D) Immunoblotting of γ-H2AX in HGC27 cells with
GLUT1 overexpression after treatment with ascorbate for 2h. β-Actin
was used as a loading control. (E-F) Mass spectra analysis of ascorbate or
DHA in the culture medium after manipulation of GLUT1 expression.
Data in B, C, E and F are presented as mean ±S.D. (n = 3). *P < 0.05
versus control. NS, non-significant.
Supplemental Figure 5: GLUT1 is overexpressed in gastric cancer
tissues and predicts poor prognosis. (A) Representative staining of
GLUT1 in two paired non-tumor tissues and tumor tissues and two paired
primary tumor and lymph node metastasis. Scale bar: 100μm.
Kaplan-Meier analysis of overall survival based on GLUT1 expression in
patients staged as I-II (B) and III-IV (C). (D-E) Kaplan-Meier analysis of
overall survival and disease free survival based on membranous GLUT1
expression in all 209 patients.
4
Supplemental Figure 6: Chemotherapeutic drugs induce ROS and
DNA damage. (A) Intracellular glutathione level of the indicated cells
treated with irinotecan (40μM, 6h), alone or in combination with
ascorbate (1mM, 1h) was measured with spectrophotometric analysis. (B)
Representative histograms (left panel) and quantification (right panel) of
ROS contents after treatment with irinotecan (40μM, 6h), alone or in
combination with ascorbate (1mM, 1h) in the indicated cells as detected
by the fluorescent probe DCF-DA. (C) Immunoblotting of γ-H2AX in
indicated cells after treatment with oxaliplatin (50μM), alone or in
combination with ascorbate (2mM) for 2h. β-Actin was used as a loading
control. (D) Immunoblotting of γ-H2AX in indicated cells after treatment
with irinotecan (50μM), alone or in combination with ascorbate (2mM)
for 2h. β-Actin was used as a loading control.
Supplemental Figure 7: Ascorbate synergize with irinotecan in
gastric cancer cells. (A) Cell viability of AGS and SGC7901 cells
treated with irinotecan alone or combined with ascorbate (1mM,2h) at
indicated concentrations was detected by MTS. (B) The combination
index (CI) of irinotecan and ascorbate treatment in AGS and SGC7901
cells was analyzed using a median dose-effect method with CalcuSyn
software (Biosoft). CI = 1 indicates an additive effect, CI < 1 indicates a
synergistic effect and CI > 1 indicates an antagonist effect. (C)
5
Representative images (left panel) and quantification (right panel) of
colony formation assays in AGS and SGC7901 cells treated with
irinotecan (2.5μM) and ascorbate (25μM). The predicted value was
calculated by multiplying the relative colony numbers in the
irinotecan-treated and ascorbate-treated sample. The combination effect is
considered additive when the observed value is equal to the predicted
value. When observed value is less than the predicted value, the
combination effect is considered as synergistic. (D) Representative
images (left panel) and quantification (right panel) of Annexin V/PI
assays in the indicated cells treated with irinotecan (50μM, 24h) and
ascorbate (2mM, 2h). (E) The volume of the tumors and the weight of
mice were measured and recorded, and a tumor growth curve was created
for each group. (F) Paraffin-embedded tumor sections were stained with
anti-Ki67 or cleaved caspase 3 antibody (scale bar: 50μm), the
proliferation and apoptosis index was quantified. Data in A, C, D, E and
F are presented as mean ±S.D. (n = 4 for A, C, D and n=6 for E, F). *P <
0.05 versus corresponding control.
Supplemental Figure 8: Ascorbate synergize with oxaliplatin or
irinotecan in MGC803 cells. (A) Representative images (left panel) and
quantification (right panel) of colony formation assays in MGC803 cells
treated with oxaliplatin (2.0μM) and ascorbate (25μM). (B)
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Representative images (left panel) and quantification (right panel) of
colony formation assays in MGC803 cells treated with irinotecan (2.5μM)
and ascorbate (25μM). The predicted value was calculated by multiplying
the relative colony numbers in the irinotecan-treated and
ascorbate-treated sample. The combination effect is considered additive
when the observed value is equal to the predicted value. When observed
value is less than the predicted value, the combination effect is considered
as synergistic. Data in A and B are presented as mean ±S.D. (n = 3). *P <
0.05 versus corresponding control.
Supplementary Figure 1
Contr
ol
Vit.(
4g/k
g)
Contr
ol
Vit.(
4g/k
g)
0
3 0
6 0
9 0
Av
era
ge
of
po
sit
ive
ce
lls
(%
)*
*
A
C
AGS SGC7901 MGC8030.0
0.3
0.6
0.9
1.2
Control
0.5mM
1mM
*
*
**
**
Rel
ativ
e A
TP L
evel
B
- + - + - +0
20
40
60
*
**
Vit.4mM AGS SGC7901 MGC803
Apopto
tic c
ells
(%
)
D
γ-H2AX
β-Actin
MGC803
Vit.
Ki67
Control Vit.(4g/kg)
Cleaved
caspase 3
Con
trol
DFO
(200
μM)
DTP
A(1
mM
)Vit.
DTP
A+V
it.
DFO
+Vit.
0
10
20
30
40
SGC7901
Ap
op
totic
ce
lls (
%)
AGS SGC7901 MGC8030
2
4
6
Control
Vit.1mM
Vit.2mM
*
*
*
*
*
*
Rela
tive
DC
F-D
A leve
l
AGS SGC79010
20
40
60
80
Control
Vit.
Vit.+NAC
*
*
Vit. + Catalase*
*
Apo
ptot
ic c
ells
(%
)
AGS SGC79010
3
6
9
12
15
18
21
Control
Vit.Vit.+NAC
**
Vit.+ Catalase
*
*
Rel
ativ
e ca
spas
e 3/
7 ac
tivity
Supplementary Figure 2
A B
C D
E F
- + - + - +
0.0
0.3
0.6
0.9
1.2
** *
Vit.
AGS SGC7901
MGC803
Rela
tive N
AD
PH
/NA
DP
+ le
vel
Con
trol
RBC
(50%
HCT)
Vit.
RBC
(50%
HCT)+
Vit.
0
2
4
6
8
10 SGC7901 *
Ap
op
totic
ce
lls (
%)
NSNS
A
Supplementary Figure 3
GES1
HGC27
SGC79
01
MGC80
3AGS
0.0
0.5
1.0
1.5 * **
Rela
tive
glu
cose u
pta
ke
GES1
HGC27
MGC80
3
SGC79
01AGS
0.0
0.3
0.6
0.9
1.2
1.5
1.8
* **
Rela
tive
lacta
te p
roduction
0
4
8
12
16
44
48
Rela
tive
gene e
xpre
ssio
n
AGS
HGC27
MGC803
SGC7901
GES1
LDH-A PKM2 PFK1 Aldolase HK2
C D
E F
B
G
- + - + - +0.0
0.3
0.6
0.9
1.2
Vit.
HGC27
Rela
tive
leve
l
DCF-DA GSH/GSSG ATP
γ-H2AX
Vit.
HGC27
β-Actin
GES1
HGC27
MGC80
3
SGC79
01AGS
0
1
2
320
22
24
26
28
30
Rela
tive
G
lut1
exp
ressio
n
GES1
HGC27
MGC80
3
SGC79
01AGS
0
5
10
15
20
25
Rela
tive
G
lut3
exp
ressio
n
GES1
HGC27
MGC80
3
SGC79
01AGS
0
1
2
3
4
Rela
tive
G
lut4
exp
ressio
n
GES1
HGC27
MGC80
3
SGC79
01AGS
0
2
4
6
Rela
tive
S
VC
T2 e
xpre
ssio
n
H I
SVCT1
β-Actin
SVCT2
Glut3
Glut4
J
GES1
HGC27
MGC80
3
SGC79
01AGS
0
2
4
6
8
10
Rela
tive S
VC
T1 e
xpre
ssio
n
A
- +
Empty
- +Vit.
γ-H2AX
β-Actin
Glut1
HGC27
- + - + - +0
1
2
3
Glut1shRNA
AGS SGC7901
MGC803
* * *
*
EV Glut1
HGC27
Rela
tive
Glu
t1
fluore
scence inte
nsity
Supplementary Figure 4
B C
D
Empty Glut1
HGC27MGC803
NC
M
sh
AGS
C
SGC7901
Glut1
Na+/K+
ATPase
Vinculin
M C
NC
M
sh
C M C
NC
M
sh
C M C M C M C
- + - +0
50
100
150
*
Vit.
Empty
Glut1
NS
Rel
ativ
e co
lony
num
ber
- + - + - +
0.0
0.5
1.0
1.5
Glut1shRNA
AGS SGC7901
MGC803
** *
EV Glut1
*
HGC27
Rela
tive
ascorb
ate
in m
ediu
m
- + - + - +
0.0
0.5
1.0
1.5
Glut1shRNA
AGS SGC7901
MGC803
* * *
EV Glut1
*
HGC27
Rela
tive
ascorb
ate
in m
ediu
m
E
F
B C
Supplementary Figure 5
0 30 60 900
20
40
60
80
100
Glut1 high (n=18)
Glut1 low (n=62)
P=0.023
Stage III-IV
Months
Perc
ent surv
ival
0 30 60 900
20
40
60
80
100
Glut1 high (n=37)
Glut1 low (n=92)
P<0.001
Stage I-II
Months
Perc
ent surv
ival
A
Normal Tumor
pair#1
Normal Tumor
pair#2
Tumor Ln Metastasis Tumor Ln Metastasis
D E
0 30 60 9020
40
60
80
100
Glut1 high (n=34)
Glut1 low (n=175)
P=0.002
Stage I-IV
Months
Perc
ent surv
ival
0 30 60 9050
60
70
80
90
100
Glut1 high (n=34)
Glut1 low (n=175)
P=0.0073
Stage I-IV
Months
Dis
ease f
ree S
urv
ival,%
AGS SGC79010.0
0.3
0.6
0.9
1.2
1.5
1.8
*
*
**
*
*
Combination
Control
Vit.
CPT11
Rela
tive
GS
H/G
SS
G leve
l
AGS SGC79010
2
4
6
8
10
Control
Vit.
CPT11
Combination
**
**
Rela
tive
DC
F-D
A leve
l
AControl1mM
40μM
Comb.
AGS
Co
un
ts
DCF-DA
Control 1mM
40μM
Comb.
SGC7901
C
Supplementary Figure 6
B
D
γ-H2AX
β-Actin
-
AGS SGC7901
+
+
--+-+ +-- +
Vit. 2mM
L-OHP, 50μM
- + +-
γ-H2AX
β-Actin
-
AGS SGC7901
+
+
--+-+ +-- +
Vit. 2mM
CPT11, 50μM
- + +-
SGC7901
0 2.5 5 10 20 400
20
40
60
80
100CPT11CPT11+Vit.(1mM)
**
*
**
*
L-OHP(μM),72h
0.0 0.2 0.4 0.6 0.80.0
0.2
0.4
0.6
0.8
1.0SGC7901
Fractional Effect
0.0 0.2 0.4 0.6 0.80.0
0.2
0.4
0.6
0.8
1.0AGS
Fractional Effect
CI
AGS
0 2.5 5 10 20 400
20
40
60
80
100
CPT11+Vit.(1mM)
*
**
**
*CPT11
L-OHP(μM),72h
Via
bili
ty (
%)
A
C
D
AGS SGC79010
20
40
60
80
Control
Vit.
CPT11
Combination
**
**
Apopto
tic c
ells
(%
)
Supplementary Figure 7
AG
S
Control
Vit.25μM
CPT11,2.5μM
CombinationAGS SGC7901
0
50
100
Control
Vit.
CPT11
Combination
*
*
Predicted
**
*
*
Rela
tive
colo
ny n
um
ber
SG
C7
90
1Control CPT11,2.5μM
Vit.25μM Combination
B
Tu
mo
r w
eig
ht(
g)
Contr
ol
Vit.
CP
T11
Com
bin
ation
0 .0
0 .5
1 .0
1 .5
2 .0 **
*
D a y s
Tu
mo
r V
olu
me
(m
m3)
0 3 6 9 1 2 1 5 1 8 2 1 2 4 2 7 3 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0C o n tro l
V it.
C P T 1 1
C o m b in a tio n *
*
0 5 1 0 1 5 2 0 2 5 3 0
1 2
1 4
1 6
1 8
2 0
2 2
D a y s
We
igh
t(g
)
C P T 1 1
V it .
C o n tro l
C o m b in a tio n
E
F
C o n t ro l V it . C P T1 1 C o m b in a t io n
0
3 0
6 0
9 0
Av
era
ge
of
po
sit
ive
ce
lls
(%
)
*
*
K i-6 7Control Vit. CPT11 Combination
Ki6
7C
lea
ve
d
ca
sp
ase
3
C o n t ro l V it . C P T1 1 C o m b in a t io n
0
3 0
6 0
9 0 **
C le a ve d
c a s p a s e 3
3.5%
12.0%
13.5%
41.0%
SG
C7
90
1
Control
Vit.2mM
CPT11,50μM
AG
S
Combination
1.6%
18.3%
31.4%
69.2%
Annexin-V
Pro
pri
um
iod
ide
Vit.2mM Combination
Control CPT11,50μM
Control
Vit.25μM
CPT11, 2.5μM
Combination
Control
Vit.25μM
L-OHP, 2μM
Combination
MGC8030
50
100
Control
Vit.
CPT11
Combination
*
*
Predicted
*
Rela
tive
colo
ny n
um
ber
MGC8030
50
100
Control
Vit.
CPT11
Combination
*
*
Predicted
*R
ela
tive
colo
ny n
um
ber
A
B
Supplementary Figure 8
Supplemental Table S1. Primers sequence for qPCR
Gene Sequence (5'-3')
Glut1-F TCCCTGCAGTTTGGCTACAA
Glut1-R AAGGCCAGCAGGTTCATCAT
HK2-F AGCACCTGTGACGACAGCAT
HK2-R ATCACATTTCGGAGCCAGGT
LDH-A -F GGTGTGAATGTGGCAGGTGT
LDH-A -R ACCATTGTTGACACGGGATG
PFK1-F AGGCTCCATTCTTGGGACAA
PFK1-R TGACCATGGGGACACAGAAC
PKM2-F GGCCAGGGAGGGACTTTTAT
PKM2-R TCACAGGCCTTCATTCGCTT
Aldolase -F CACTCGTACCCAGCCCTTTC
Aldolase -R ACGCCTCCAATGCACTTTTT
SVCT1 -F TTCCAGGCACGAACCGATG
SVCT1 -R AGTGGCGCTGAACATTCCC
SVCT2 -F CTTCACTCTTCCGGTGGTGAT
SVCT2 -R TTTCCGTAGTGTAGATCGCCA
Glut3 -F GCTGGGCATCGTTGTTGGA
Glut3 -R GCACTTTGTAGGATAGCAGGAAG
Glut4 -F TGGGCGGCATGATTTCCTC
Glut4 -R GCCAGGACATTGTTGACCAG
β-Actin -F CATGTACGTTGCTATCCAGGC
β-Actin -R CTCCTTAATGTCACGCACGAT
Supplemental Table S2. The correlation between clinicopathological parameters and Glut1
expression
*P < 0.05.
Glut1 expression
P Low, n(%) High, n(%)
Age
﹤60 105 (77.8) 30(22.2) 0.074
≥60 49(66.2) 25(33.8)
Gender
Male 96(70.1) 41(29.9) 0.069
Female 58(80.6) 14(19.4)
Tumor size
﹤5cm 100(80.6) 24(19.4) 0.007*
≥5cm 54(63.5) 31(36.5)
Differentiation status
Well or Moderate 26(53.1) 23(46.9) 0.000*
Poor and others 128(80.0) 32(20.0)
Lymph node invasion 0.727
Absent 109(72.7) 41(27.3)
Present 45(76.3) 14(23.7)
Venous invasion 0.084
Absent 81(79.4) 21 (20.6)
Present 73(68.2) 34(31.8)
Perineural invasion 0.003*
Absent 38(59.4) 26(40.6)
Present 116(80.0) 29(20.0)
TNM stage 0.338
I-II 92(71.3) 37(28.7)
III-IV 62(77.5) 18(22.5)
Supplemental Table S3. Univariate and multivariate analyses of various potential
prognostic factors in GC patients
Univariate analysis Multivariate analysis
HR (95% CI) P HR (95% CI) P
Age (<60/≥60) 0.71-1.81 0.611 - -
Gender (male/female) 0.69-1.76 0.690 - -
Differentiation
(well, moderate/poor)
0.83-2.65 0.184 - -
Tumor size (≥5cm/<5cm) 0.82-2.02 0.269 - -
Lymph node invasion
(present/absent)
Venous invasion
(present/absent)
Perineural invasion
(present/absent)
0.80-2.07
1.34 -3.57
1.25-4.14
0.306
0.002*
0.007*
-
0.66-1.91
0.97-3.65
-
0.671
0.062
TNM Stage(III-IV/I-II) 2.54-6.55 0.000* 2.23-6.40 0.000*
Glut1 protein (high/low) 1.21-3.07 0.006* 1.70-4.66 0.000*
HR: hazard ratio; CI: confidence interval; *P < 0.05.