Supplemental Figure 1: Ascorbate induces apoptosis and inhibits … · 2018-02-06 · Supplemental...

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)

6

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

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Con

trol

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P=0.023

Stage III-IV

Months

Perc

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Months

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pair#1

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D E

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Months

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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.

Supplemental Figure 1: Ascorbate induces apoptosis and inhibits … · 2018-02-06 · Supplemental...

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