Figure 1.
Cycling hypoxia triggers ROS production via Nox4 in glioblastoma cells.
GBM8401 and U87 cells were treated with cycling hypoxic stress for 4 h in the absence or presence of Nox4 siRNA or 10 µM diphenyleneiodonium chloride (DPI), and the levels of intracellular ROS (A), H2O2 (B), Nox4 mRNA (C), and Nox4 protein (D) were evaluated by H2DCHFDA reagent, Amplex Red assay, Q-PCR, and western blotting, respectively. Each bar represents the mean ± standard deviation of triplicate measurements. * p<0.01 compared to normoxia. # p<0.01 compared to cycling hypoxia.
Figure 2.
Nox 4 knockdown and a antioxidant compound suppress cycling hypoxia-induced ROS levels in glioblastoma xenografts.
(A) Regulation of Nox4 by Dox-inducible shRNA. GBM8401-Luc cells were infected with Tet-regulable lentiviral vectors encoding Nox4 shRNAs. The infected cells were treated with or without Dox for 24 h and harvested for western blot analysis. (B) Immunohistochemical analysis of Nox4 in GBM8401-Luc xenografts with or without conditional knockdown of Nox4 under cycling hypoxic stress. Original magnification, ×200. Bar, 100 µm. (C) In vivo optical imaging of GBM-bearing mice injected with L-012. (D) Quantitative data obtained from in vivo optical imaging of ROS levels in GBM xenografts with or without Dox or Tempol following in vivo cycling hypoxic stress. * p<0.01 compared to normoxia. # p<0.01 compared to cycling hypoxia.
Figure 3.
Cycling hypoxia induces higher, long-term HIF-1 activation in glioblastoma cells and xenografts.
(A) Western blot analysis of HIF-1α in GBM8401 and U87 cells after cycling hypoxia. Cells were exposed to hypoxic stress, either non-interrupted or cycling, for 4 h and harvested to determine the amounts of HIF-1α protein in nuclear extracts. (B) Transcriptional activity at hypoxia response elements in GBM8401 cells after cycling hypoxic stress. GBM8401/hif-1-r cells were cultured under hypoxic stress, either non-interrupted or cycling, for 4 h and grown in normoxia for different periods, followed by measurements of reporter gene expression. (C) Kinetics of HIF-1 transcriptional activity in GBM8401/hif-1-r xenografts after cycling hypoxic stress. In vivo fluorescence imaging (FLI) was performed for GBM8401/hif-1-r tumors before hypoxic treatments and at different times after hypoxic treatments. The data represent the mean ± standard deviation of the ratio of average counts within the tumor region of interest (ROI) in GFP and DsRed signals from 6 mice.
Figure 4.
ROS is required for cycling hypoxia-induced HIF-1 activation in glioblastoma cells and xenografts.
(A) Flow cytometric analysis of HIF-1 transcriptional activity in GBM8401/hif-1-r and U87/hif-1-r cells exposed to cycling hypoxic stress with or without Tempol. In vivo microPET imaging (B) and in vivo optical imaging (C) of HIF-1 transcriptional activity in GBM8401/hif-1-r tumors with or without Tempol treatment. MicroPET imaging with [18]FHBG and in vivo optical imaging were used to determine in vivo HIF-1 signal transduction activity 24 h after in vivo cycling hypoxia treatment.
Table 1.
Quantitative data obtained from microPET imaging and in vivo optical imaging of HIF-1 transcriptional activity in GBM8401/hif-1-r tumors with or without Tempol treatment.
Figure 5.
The majority of HIF-1 signal transduction activity and Nox4 expression occurs in endogenous cycling hypoxic areas in a solid tumor.
(A) Representative images of microscopic GBM8401/hif-1-r xenografts. Upper left, fluorescence image of DsRed reporter (red), indicating tumor cell localization within the brain. Upper right, fluorescence image of Hoechst 33342 (blue) showing perfusion within the brain and tumor tissue. Lower left, fluorescence image of GFP reporter (green), demonstrating HIF-1 transcriptional activity in tumor cells. Lower middle, fluorescence image of Nox4 staining (red). Lower right, fluorescence overlay image of Hoechst 33342 (blue), GFP reporter (green), and Nox4 (red). Bar, 50 µm. (B) Scatterplots by 2-color staining with Hoechst 3342 and GFP. (C) Mean channel fluorescence of Nox4 staining was determined in cycling hypoxic cells (Hoechst 3342+ and GFP+), chronic hypoxic cells (Hoechst 3342− and GFP+), and normoxic cells (Hoechst 3342+ and GFP−) as gated in scatterplots by Hoechst 3342 and GFP staining.
Figure 6.
Cycling hypoxia promotes tumor growth via Nox4-mediated ROS in GBM xenografts.
(A) The mean normalized BLI values associated with longitudinal monitoring of intracranial tumor growth for each treatment group. Mice bearing 12-d orthotopic GBM8401-Luc xenografts were treated daily with Dox-inducible Nox4 knockdown or 100 mg/kg Tempol following in vivo cycling hypoxia treatment for 24 days. Bars report the mean ± standard deviation of measurements in 6 mice. (B) The corresponding survival curves of GBM8401-Luc xenograft-bearing mice exposed to daily treatment with Dox-inducible Nox4 knockdown or Tempol following in vivo cycling hypoxia treatment. * p<0.01 compared to normoxia.