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. 2014 Jan 23:4:3793.
doi: 10.1038/srep03793.

VSports注册入口 - HIF-1-mediated metabolic reprogramming reduces ROS levels and facilitates the metastatic colonization of cancers in lungs

Tao Zhao  1 , Yuxi Zhu  2 , Akiyo Morinibu  3 , Minoru Kobayashi  3 , Kazumi Shinomiya  3 , Satoshi Itasaka  4 , Michio Yoshimura  4 , Guozheng Guo  5 , Masahiro Hiraoka  4 , Hiroshi Harada  3
Affiliations

HIF-1-mediated metabolic reprogramming reduces ROS levels and facilitates the metastatic colonization of cancers in lungs (VSports手机版)

Tao Zhao et al. Sci Rep. .

Abstract

Hypoxia-inducible factor 1 (HIF-1) has been associated with distant tumor metastasis; however, its function in multiple metastatic processes has not yet been fully elucidated. In the present study, we demonstrated that cancer cells transiently upregulated HIF-1 activity during their metastatic colonization after extravasation in the lungs in hypoxia-independent and reactive oxygen species (ROS)-dependent manners. Transient activation induced the expression of lactate dehydrogenase A and phosphorylation of the E1α subunit of pyruvate dehydrogenase, which indicated the reprogramming of glucose metabolic pathways from mitochondrial oxidative phosphorylation to anaerobic glycolysis and lactic acid fermentation. The administration of the HIF-1 inhibitor, YC-1, inhibited this reprogramming, increased intratumoral ROS levels, and eventually suppressed the formation of metastatic lung tumors. These results indicate that HIF-1-mediated metabolic reprogramming is responsible for the survival of metastatic cancers during their colonization in lungs by reducing cytotoxic ROS levels; therefore, its blockade by HIF-1-inhibitors is a rational strategy to prevent tumor metastasis VSports手机版. .

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Figure 1
Figure 1. Transient upregulation of HIF-1 activity during the metastatic colonization of cancers in the lungs.
(a,b) Twenty-four hours after seeding EMT6 (a) and EMT6/CMVp-CLuc (a,b) cells (1 × 104 cells in 100 μL culture medium per one well of a 24-well plate), 10 μL culture medium was subjected to the CLuc assay. Means ± s.d. n = 3. **P < 0.01. (c,d) After the subcutaneous transplantation of EMT6/CMVp-CLuc cells, tumor growth and CLuc activity in the serum were monitored by measuring the tumor volume with calipers (c) and by performing the in vivo CLuc assay (d), respectively. Means ± s.d. n = 10. (e) Positive correlation between CLuc activity in the serum and the volume of subcutaneous tumor xenografts measured with calipers. (f) The relationship between CLuc activity in the serum and the number of metastatic colonies in the lungs was analyzed in 20 independent mice 10 days after the i.v. transplantation of EMT6/CMVp-CLuc cells. (g) EMT6/CMVp-CLuc/5HREp-FLuc cells (1 × 104 cells/well in 24-well plate) were cultured under normoxic (20% O2) or hypoxic (0.02% O2) conditions for 24 hours and harvested for the firefly luciferase assay. Means ± s.d. n = 3. **P < 0.01. (h) On the indicated days after the i.v. transplantation of EMT6/CMVp-CLuc/5HREp-FLuc cells (1 × 106/mouse), mice were subjected to optical in vivo imaging using the IVIS SPECTRUM system to externally monitor HIF-1 activity in metastatic lung tumors as FLuc bioluminescence (See Supplementary Fig. S4a). Ten μL of serum was simultaneously harvested from mice and subjected to the in vivo CLuc assay to monitor the volume of metastatic tumors (See Supplementary Fig. S4b). HIF-1 activity in lung metastases was divided by CLuc activity in the serum to calculate HIF-1 activity per unit of metastatic lung tumors. Means ± s.d. n = 10. *P < 0.05.
Figure 2
Figure 2. ROS-mediated transient activation of HIF-1 during the metastatic colonization of cancers in the lungs.
(a) Tumor hypoxia (pimonidazole; green) and CD31 (red) were detected on the indicated days after the i.v. transplantation of EMT6/CMVp-CLuc/5HREp-FLuc cells in the lungs with the resultant metastatic tumors (dotted line). Hoechst33342 = counter staining (blue). Bar = 50 μm. (b) On the indicated days after the i.v. transplantation of cancers, oxidative stress in methacarn-fixed paraffin-embedded sections (upper) and formalin-fixed paraffin-embedded sections (lower) of the lungs with the resultant metastatic tumors were detected by the Protein Carbonyls Immunohistochemical Staining Kit (upper) and anti-phospho-p38 antibody (lower), respectively. Bar = 500 μm (upper) and 20 μm (lower). (c–e) EMT6/CMVp-CLuc/5HREp-FLuc cells were cultured with (+) or without (−) N-acetylcysteine (NAC) under 3% oxygen conditions for 24 hours or under 3% oxygen conditions for 18 hours followed by 15% oxygen conditions for 6 hours, and were then treated with the ROS marker, DCFH-DA, for flow cytometric analysis (c), subjected to Western blotting for protein carbonyls as the markers of oxidative stress (upper) and for β-actin (lower) (d), and subjected to the firefly luciferase assay for HIF-1 activity (e). A representative data set from 3 independent experiments is shown (c). Means ± s.d, n = 3, **P < 0.01 (e). (f–h) Athymic nude mice were i.v. transplanted with a suspension of EMT6/CMVp-CLuc/5HREp-FLuc cells (1 × 106/mouse) and treated with NAC (1,000 mg/kg) or saline (control) twice a day from 1 to 5 days after the transplantation. Six days after the transplantation, mice were subjected to the in vivo CLuc assay in the serum for tumor volume (f) and to the optical in vivo imaging experiment for HIF-1 activity (g). HIF-1 activity in lung metastases was divided by the CLuc activity in the serum to calculate HIF-1 activity per unit of metastatic lung tumors (h). Means ± s.d. n = 10. *P < 0.05. **P < 0.01. NS = not significant.
Figure 3
Figure 3. HIF-1-mediated metabolic reprogramming during the metastatic colonization of cancers in the lungs.
(a) Athymic nude mice were i.v. transplanted with a suspension of the stable transfectant, EMT6/CMVp-Cluc/5HREp-Luc (1 × 106/mouse). Tumor-bearing mice were treated with saline (-), NAC (1,000 mg/kg/administration, 2 injections/day from days 1 to 6), or YC-1 (30 mg/kg daily from days 3 to 6). On the indicated days after the i.v. transplantation, LDHA (green) and p-PDH-E1α (red) were detected in the lungs with the resultant metastatic tumors (dotted line). Hoechst33342 = counter staining (blue). Bar = 50 μm. (b–d) Athymic nude mice were i.v. transplanted with a suspension of EMT6/CMVp-CLuc/5HREp-FLuc cells, and treated with or without the HIF-1 inhibitor, YC-1 (30 mg/kg daily) from 3 to 6 days after the transplantation of cancer cells. Six days after the transplantation, mice were subjected to the in vivo CLuc assay for tumor volume (b) and to the optical in vivo imaging experiment for HIF-1 activity (c). HIF-1 activity in lung metastases was divided by CLuc activity in the serum to calculate HIF-1 activity per unit of metastatic lung tumors (d). Means ± s.d. n = 10. *P < 0.05. **P < 0.01. NS = not significant.
Figure 4
Figure 4. HIF-1-mediated metabolic reprogramming reduced ROS levels and increased the survival of metastatic cancers.
Athymic nude mice were i.v. transplanted with a suspension of the stable transfectant, EMT6/CMVp-CLuc/5HREp-FLuc cells (1 × 106/mouse), and treated with or without the HIF-1 inhibitor, YC-1 (30 mg/kg daily) from 3 to 6 days after the transplantation of cancer cells. (a) Lungs with the resultant metastatic tumors were surgically excised 7 days after the transplantation, which was 1 day after the peak in HIF-1 activity and ROS levels (See Fig. 1h,2b), and ROS was detected in methacarn-fixed paraffin-embedded sections (upper) and formalin-fixed paraffin-embedded sections (lower) by the Protein Carbonyls Immunohistochemical Staining Kit and anti-phospho-p38 antibody, respectively. Bar = 500 μm (upper) and 20 μm (lower). (b) Ten days after the transplantation, mice were subjected to the in vivo CLuc assay in the serum for tumor volume. (c) Lungs with the resultant metastatic tumors were surgically excised 10 days after the transplantation, treated with Bouin's solution, and the number of colonies was externally counted. Means ± s.d. n = 10. *P < 0.05. (d) Representative images of the lungs are shown.
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