Grant support was provided by the National Science Council of the Republic of China (Grant No. 102-2314-B-039-029-MY3) and grants CMU101-S-17 and
CMU103-BC-6 from China Medical University.
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Figure legends
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Figure 1. Tumor hypoxia induces Livin expression. (A) Time course of Livin mRNA expression after exposure of GBM8401 cells to <1% O2. Livin mRNA (B) and protein (C) levels in U251, U87 and GBM8401 cells at 24 hours after normoxic (N.) and hypoxic (H.) treatment (<1% O2). (D) Representative images of microscopic
GBM8401/hif-1-r. Top left, fluorescence overlay image of perfusion marker, Hoechst 33342 (blue), and HIF-1 activation labeling, GFP reporter (green), indicating areas with normoxia (Hoechst 33342+ and GFP-), chronic hypoxia (Hoechst 33342- and GFP+) and cycling hypoxia (Hoechst 33342+ and GFP+). Top right, fluorescence overlay image of Hoechst 33342 (blue), GFP reporter (green), and Livin (red). White box 1 and 2 indicate cycling hypoxic area and chronic hypoxic area, respectively.
Bottom left and right, magnification of white box 1 and 2. While color indicates the localization of Hoechst 33342, GFP and Livin. Yellow color represents the co-localization of GFP and Livin. (Magnification: Upper, 100X; Lower, 200X) (E) Livin and VEGF mRNA levels in normoxic cells (Hoechst 3342+ and GFP-), chronic
hypoxic cells (Hoechst 3342- and GFP+) and cycling hypoxic cells (Hoechst 3342+ and GFP+) isolated from disaggregated GBM8401/hif-1-r and U87/hif-1-r xenografts.
Error bars denote the standard deviation within triplicate experiments. *P < 0.01 compared to normoxia. # P <0.01 compared to chronic hypoxia (F)
Immunofluorescence imaging of Livin and HIF-1α expression in primary GBMs.
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Upper panels show the staining of DAPI (blue), HIF-1α (green), and Livin (red).
Lower panels demonstrate the overlay image of DAPI (blue), HIF-1α (green), and Livin (red) and indicate the magnification of while boxes. Yellow color indicates the co-localization of HIF-1α and Livin. (Magnification: Upper, 100X; Lower, 100X, 200X and 400X)
Figure 2. Hypoxia-induced Livin expression is dependent on HIF-1α. (A)
Verification of HIF-1α or HIF-2α knockdown by HIF-1α or HIF-2α shRNAs. Livin mRNA (B) and protein (C) levels in GBM8401 and U87 with or without HIF-1α or HIF-2α knockdown at 24 hours after hypoxic treatment (<1% O2). (D) Luciferase reporter assay shows that HIF-1α shRNA inhibited the hypoxia-induced
transcriptional activation of Livin in U251, U87 and GBM8401 glioblastoma cells.
Error bars denote the standard deviation within triplicate experiments. *P < 0.001 compared to normoxia. # P <0.001 compared to scramble (Scr.) shRNA.
Figure 3. HIF-1α binds directly to the Livin promoter to regulate its expression. (A) Graphic representation of the putative Livin promoter. Two putative hypoxia response elements (HREs) were identified. (B) Luciferase reporter plasmids carrying the wild type or mutant Livin promoter regions were co-transfected with the Renilla luciferase 628
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reporter plasmid into GBM8401 cells; and the cells were treated with or without hypoxia (<1% O2) for 24 hours. (C and D) Chromatin immunoprecipitation followed by real-time PCR (ChIP-qPCR) assay of HIF-1α binding in Livin promoter in
response to hypoxia (<1% O2) for 24 hours. Results are expressed as percentage of input. Error bars denote the standard deviation among triplicate experiments. *P <
0.001 compared to control or normoxia. # P <0.001 compared to vehicle or wild type.
Figure 4. Hypoxia-mediated Livin expression results in antiapoptosis. Verification of Livin knockdown by Tet-regulatable lentiviral knockdown system in mRNA (A) and protein (B) levels. The lentiviral infected cells were treated for 48 hours with Dox (0.04 μg/mL) to induce Livin knockdown. Error bars denote the standard deviation among triplicate experiments. *P < 0.001 compared to no Dox treatment. Caspase-3 activities (C) and percent of apoptotic cells (D) in GBM8401 and U87 cells cultured in normoxia, non-interrupted (Ni.) hypoxia (<1% O2) and cycling (Cy.) hypoxia (<1%
O2) with or without Livin knockdown in response to staurosporine (STA) treatment.
Cells were pretreated for 48 hours with Dox (0.04 μg/ml) to induce Livin knockdown and exposed to hypoxic stresses before STA treatment. Error bars denote the standard deviation among triplicate experiments. *P < 0.01 compared to control. # P <0.05 compared to normoxia with STA treatment.
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Figure 5. Hypoxia-mediated Livin expression promotes radioresistance and
chemoresistance. (A) Radiation cell survival curves for GBM8401 cells. Cells were pretreated for 48 hours with Dox to induce Livin knockdown and exposed to hypoxic stress (<1% O2), either non-interrupted (Ni.) or cycling (Cy.) hypoxia, before
irradiation. (B) Cytotoxicity assay of Livin knockdown revealed increased
cytotoxicity of cells to TMZ and suppressed hypoxia-mediated TMZ resistance in GBM8401 and U251 glioblastoma cells. *P < 0.01 compared to normoxia. # P <0.01 compared to untreated Dox groups. (C) Sequences of TAT-Lp15 amino acids and K22 control peptide. (D) Visualization of intracellular accumulation of TAT-Lp15-dansyl or TAT-K22-dansyl (10 μM) at 60 minutes after incubation to
GBM8401 cells. (E) Time course of TAT-Lp15-dansyl (10 μM) fluorescence after incubation to GBM8401 cells. Fluorescence intensities were determined by flow cytometry. Surviving fraction (F) and cell viability (G) for U251, U87 and GBM8401 cells with or without TAT-K22 or TAT-Lp15 (30μM) and receiving hypoxic stress (H) (<1% O2) followed by 4-Gy irradiation (R) or 100-μM TMZ (T). Error bars denote the standard deviation among triplicate experiments. *P < 0.001 compared to control. # P <0.001 compared to radiation or TMZ alone. ※P <0.001 compared to hypoxia combined radiation or TMZ groups.
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Figure 6. Livin blockage enhances the efficiency of radiation plus TMZ treatment in glioblastoma xenografts. (A) Detection of TAT-Lp15-dansyl and TAT-K22-dansyl in the cortex of nude mouse brain 1 hour after intravenous injection. Bars=100 μm (B) Bioluminescent images from control and treated animals including the TAT-K22 or TAT-Lp15 treatment, combination of TMZ (T) and radiation (R) treatment,
pretreatment of TAT-K22 plus the combination of TMZ and radiation treatment and pretreatment of TAT-Lp15 plus the combination of TMZ and radiation treatment on day 20 after tumor implantation. (C) The mean normalized BLI values associated with longitudinal monitoring of intracranial tumor growth for each treatment group. Error bars denote the standard deviation among 9 mice per group. (D) The corresponding survival curves of GBM8401 xenograft-bearing mice for each treatment group. (E) Representative sections showing apoptotic cells measured by the TUNEL assay of GBM8401 xenografts at 2 days after treatments. Each mean is based on 15
measurements (5 measurements for each of three tumors).
(F) The percentages of apoptotic cells on cross-sections. *P < 0.01 compared to control. #P <0.001 compared to control TAT-K22 peptide. (G) The percentages of apoptotic cells in tumor hypoxic cells within GBM8401 xenografts determined by pimonidazole/annexin V doubling staining flow cytometry assay. *P < 0.001 685
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compared to control. #P <0.01 compared to control TAT-K22 peptide. Surviving fraction (H) and colony images (I) in excised GBM8401 and U251 xenografts after treatments. Error bars denote the standard deviation among triplicate experiments. *P
< 0.001 compared to combination of TMZ and radiation treatment.
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