Article Figures & Data
Figures
- Figure 1.
Andro sensitizes human cancer cells to TRAIL-induced apoptosis. A, TRAIL-induced apoptosis in human cancer cells. HepG2, HCT116, and HeLa cells were treated with indicated concentrations of TRAIL for 24 h. B, Andro-induced apoptosis in human cancer cells. Cancer cells were treated with indicated doses of Andro for 24 h. C, sensitization effect of Andro on TRAIL-induced apoptosis. HepG2, HCT116, and HeLa were pretreated with the indicated concentration of Andro for 2 h followed by treatment with a subtoxic concentration of TRAIL (10 ng/mL for HepG2 and 1 ng/mL for HCT116 and HeLa) for another 12 h. The percentage of apoptosis was determined using DAPI staining. Mean ± SD of three independent experiments. Representative images of HepG2 cells with various treatments were photographed using a normal light microscope and an inverted fluorescence microscope. Magnification, ×200.
- Figure 2.
Andro enhances the caspase cascade triggered by TRAIL. A, Andro promotes TRAIL-induced caspase activation. HepG2 cells were pretreated with 15 μmol/L Andro for 2 h followed by treatment with TRAIL (10 ng/mL) for indicated periods. Cell lysates were collected and subjected to Western blot for detecting the cleavage of caspase-8, caspase-3, and PARP. B, caspase inhibitors block PARP cleavage induced by Andro and TRAIL. C, caspase inhibitors block apoptosis induced by Andro and TRAIL. B and C, HepG2 cells were pretreated with Z-IETD-CHO (25 μmol/L), Z-DEVD-CHO (25 μmol/L), or Z-VAD-FMK (25 μmol/L) for 30 min followed by combined treatment of Andro (15 μmol/L) for 2 h and then TRAIL (10 ng/mL) for another 12 h. Cells were collected for measurement of PARP cleavage by Western blot (B) or determination of cell death using DAPI staining (C). Mean ± SD of three independent experiments. Magnification, ×200.
- Figure 3.
Andro up-regulates DR4 transcription. A, time-dependent up-regulation of DR4 protein level by Andro. HepG2 cells were treated with Andro (15 μmol/L) for 2 h followed by TRAIL (10 ng/mL) for indicated periods. Cell lysates were collected for Western blot to detect protein expression level of various TRAIL death receptors. B, effects of Andro on the cell surface expression of DR4 and DR5. HepG2 cells were incubated with Andro (15 μmol/L) for 6 h and the cell surface expression of DR4 and DR5 proteins was analyzed by flow cytometry. X axis, fluorescence intensity; Y axis, relative number of cells. Representative of three independent experiments. C, effects of Andro and TRAIL on DR4 and DR5 mRNA level. HepG2 cells were treated with Andro (15 μmol/L) for 2 h followed by TRAIL (10 ng/mL) for indicated periods. The mRNA levels of DR4 and DR5 were measured by reverse transcription-PCR. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. D, DR4 blocking antibody suppresses the cleavage of caspase-8 and PARP induced by Andro and TRAIL. HepG2 cells were pretreated with DR4 or DR5 blocking antibodies (10 μg/mL) for 30 min followed by pretreatment with Andro (15 μmol/L × 2 h) and then TRAIL (10 ng/mL) for another 12 h. At the end of treatment, cell lysates were collected for Western blot. E, DR4 blocking antibody prevents Andro-sensitized apoptosis. HepG2 cells were treated as indicated in D. At the end of treatment, cell death was determined using DAPI staining. Magnification, ×200.
- Figure 4.
p53 is required for DR4 up-regulation and enhanced apoptosis by Andro. A, Andro promotes p53 protein accumulation. HepG2 cells were treated with Andro (15 μmol/L) for 2 h followed by TRAIL (10 ng/mL) for indicated periods. p53 and DR4 protein levels were detected by Western blot. B, p53 knockdown prevents Andro-induced DR4 up-regulation. HepG2 cells were transfected with scrambled siRNA or p53 siRNA for 24 h. Cells were treated with Andro (15 μmol/L) for 6 h. At the end of treatment, cells were collected for detection of p53 and DR4 by Western blot. C, p53 knockdown suppresses apoptosis induced by Andro and TRAIL. After p53 knockdown, HepG2 cells were treated with Andro (15 μmol/L) for 2 h followed by TRAIL (10 ng/mL) for 12 h. Cells were assessed using DAPI staining. Representative of three independent experiments. Magnification, ×200.
- Figure 5.
Andro sensitizes TRAIL-induced apoptosis in p53 wild-type cancer cells but not in p53-deficient cancer cells. A, HepG2 and Hep3B cells were pretreated with Andro (15 μmol/L) for 2 h followed by TRAIL (10 ng/mL) for another12 h. The percentage of apoptosis was determined using DAPI staining. Mean ± SD of three independent experiments. Magnification, ×200. B, HCT116 p53 wild-type cells and HCT116 p53 knockout cells were pretreated with Andro (15 μmol/L) for 2 h followed by TRAIL (1 ng/mL) for another12 h. The percentage of apoptosis was determined using DAPI staining. Mean ± SD of three independent experiments. Magnification, ×200. C, Andro fails to enhance DR4 level in HCT116 p53 knockout cells. HCT116 p53 wild-type cells and HCT116 p53 knockout cells were treated with Andro (15 μmol/L) for 6 h or 12 h. At the end of treatment, cells were collected for detection of p53, DR4, and p21 using Western blot.
- Figure 6.
Andro promotes JNK activation, p53 stabilization, and DR4 up-regulation in ROS-dependent manner. A, Andro induces JNK activation. HepG2 cells were treated with Andro (15 μmol/L) for 2 h followed by TRAIL (10 ng/mL) for indicated periods. At the end of treatment, cell lysates were collected for the detection of protein level of phospho-JNK, total JNK, phospho-p53 Thr81, p53, DR4, and α-tubulin using Western blot. B, NAC and SP600125 abrogate Andro-induced JNK activation, p53 phosphorylation, and stabilization. HepG2 cells were pretreated with NAC (2.5 mmol/L for 30 min) or SP600125 (20 μmol/L for 30 min) followed by the treatment of Andro (15 μmol/L × 2 h) and then TRAIL (10 ng/mL) for another 6 h. C, NAC and SP600125 prevent Andro-induced DR4 up-regulation. Cells were first pretreated with NAC and SP600125 as described in B followed by Andro (15 μmol/L × 2 h) and then TRAIL (10 ng/mL) for another 3 h (for detection of DR4 mRNA level) and 6 h (for detection of DR4 protein level). D, NAC prevents Andro-induced cell surface expression of DR4. HepG2 cells were pretreated with NAC as described above followed by Andro (15 μmol/L) for 6 h. The cell surface expression of DR4 was measured using flow cytometry as described in B. Representative of three independent experiments. E, NAC and SP600125 inhibit apoptosis induced by Andro and TRAIL. HepG2 cells were treated with Andro (15 μmol/L × 2 h) followed by TRAIL (10 ng/mL) for another 12 h. The percentage of apoptosis was measured with DAPI staining. Mean ± SD of three independent experiments. Magnification, ×200.
Additional Files
Supplementary Material, Zhou et al
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Volume 7, Issue 7
