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.
