Summary
Moderate swimming boosts dopaminergic activity and suppresses growth and metastasis of liver
cancer in mice model via activation of dopamine receptor 2 (DR2) signaling, while overload
swimming has the opposite effect. Together, these findings elucidate a novel mechanism
underlying protective effect of moderate exercise against cancer with role of dopamingric system
and suggest agonist of DR2 may have suppressive effect on liver cancer.
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Supplementary Results
Fig. s1. Swimming training impacts on body weight of C57BL/6 mice.
After 6 weeks swim training (including 3 weeks before inoculation), compared to control, body
weight was decreased in swim 8 min/day group (*p < 0.01), dramatically in group of 16 min/day,
swim 32 min/day (**p < 0.001).
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Fig. s2. Pathological confirmation of liver cancer metastasis in the lung from C57BL/6 mouse
injected with Hep1-6-GFP liver cancer cells by tail vein (H&E staining; scale bar = 500μm; Lung
metastasis foci indicated by black arrow).
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Fig. s3. Serum DA levels increased with swimming training periods.
The impact of moderate swimming on serum DA levels were detected with ELIDA kit. Serum DA
(mean ± SD) increased in the first 3 days, gradually fell to baseline levels after 7 days of
swimming, and then the DA levels gradually increased after 14 days swimming. The serum DA
levels were significantly increased at 28 days (p = 0.025) and even more dramatically increased
from 42 days to 70 days (all p < 0.01).
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Fig. s4. Expression of DRD1-5 in normal human liver cell, human cancer cells, and murine liver
cancer cells.
(A) RT-PCR for expression of DRD1-5 in normal human liver cells (L02), human cancer cells
(SMMC-7721 and HCC-LM3), and murine liver cancer cells (Hepa1-6 and H22). (B) Western
blots showed DR1 and DR2 expression in normal human liver cells (L02), human cancer cells
(SMMC-7721 and HCC-LM3), and murine liver cancer cells (Hepa1-6 and H22). (C)
Immunofluorescence showed both DR1 and DR2 expression on the membrane of Hepa1-6 and
HCC-LM3 cells in vitro.
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Fig. s5. DA or MS suppressed growth and lung metastasis of liver cancer by acting on DR2.
(A) Mice that were subcutaneously injected with Hepa1-6 hepatomas were exposed to MS or MS
+ domperidone, the DR2 antagonist. Tumors from each of the 6 mice in the group were examined.
(B) Lung metastasis foci from C57BL/6 mouse on day 42 after injection with Hepa1-6-GFP by tail
vein (n=6 in each group), one sample from a control, moderate swimming, and moderate
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swimming + domperidone group are shown (n=6 in each group). (C) Tumor weights (mean ± SD)
from C57BL/6 mice with subcutaneous Hepa1-6 hepatomas that were subjected to over-load
swimming or over-load swimming + bromocriptine were compared with controls (n=6 in each
group). Bromocriptine
canceled the enhancing effect of over-load swimming on growth of
hepatomas. (D) Lung metastasis from C57BL/6 mice that had been injected with Hepa1-6-GFP
cells via tail vein and subjected to over-load swimming or over-load swimming + bromocriptine
(n=6 in each group). Bromocriptine canceled the suppressive effect of moderate swimming on
lung metastasis. Data are presented as mean ± SD. (E) Bromocriptine suppressed growth of
othortopicly implanted HCC-LM3-GFP in nude mice in a comparison with controls (n=6 in each
group). (F) Two representative lung metastasis foci of HCC-LM3-GFP day 42 after othortopic
implantation (n=6 in each group).
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Supplementary Table 1. The dopamine levels in prefrontal cortex, serum and subcutaneous
tumor of four groups.
DA level
Control
Swim 8 min/d
Swim 16 min/d
Swim 32 min/d
Prefrontal cortex (ng/ml)
35.6 ± 13.2
58.3 ± 17.6**
39.6 ± 14.2*
23.5 ± 12.4*
Serum
(pg/ml)
257.8 ± 72.1
433.9 ± 102.1**
234.2 ± 82.3*
201.3 ± 95.4*
Tumor tissue
(pg/ml)
235.3 ± 60.3
361.6 ± 95.8**
210.2 ± 57.4*
183.7 ± 52.6*
*p < 0.05, **p < 0.01.
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Supplementary Table 2. Primer sequences and Tm used for RT-PCR.
Tm
Gene
Forward primer
Reverse primer
Amplicon
(°C)
(bp)
human DRD1
5' GAA GGC ATT TTC AAC CCT C 3'
5' TCA CCG TCT CTA TGG CAT T 3'
79.9
71
human DRD2
5' TCA GAG GTT CTT TGA GGG A 3'
5' AAG GGA AAC AGG AGA GAG G 3'
82.2
96
human DRD3
5' TGT CCT TCT ACC TGC CCT T 3'
5' TTT CCG TCT CCT TTG TTT C 3'
80.6
80
human DRD4
5' CGC CCT CAA CCC CGT CAT 3'
5' CTC AGC AGC AGG CAC GCA 3'
87.0
83
human DRD5
5' TAG AAA CCT CAC CCC ATT G 3'
5' AAG GAC CCA GCA CCA TAC T 3'
82.5
135
human TGF-β1
5' ACC TGA ACC CGT GTT GCT C 3'
5' GAA CCC GTT GAT GTC CAC T 3'
88.8
286
mouse Drd1
5' TCC TCC CTG AAC CCC ATT A 3'
5' CCC GTT GTT GTT GAT GCT T 3'
81.6
132
mouse Drd2
5' CCC CAC TAC AAC TAC TAT G 3'
5' CAT TGC CAA AGA CGA TGA T 3'
79.4
64
mouse Drd3
5' ATC ATC TTT GGC AAC GGT C 3'
5' GTG GTC TGT AGG GCT CGC T 3'
82.4
62
mouse Drd4
5' CAC CGC CTC CAT CTT CAA C 3'
5' CCA CGA ACC TGT CCA CGC T 3'
82.8
50
mouse Drd5
5' TCT CAA ATC TCT CCA ACG A 3'
5' AAC CTC CTC CTC ACA GTC A 3'
83.5
78
mouse Tgfb1
5' AAG GAC CTG GGT TGG AAG T 3'
5' CGG GTT GTG TTG GTT GTA G 3'
86.7
135
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