Journal: Oncology reports

Article Title: AZD8055 induces cell death associated with autophagy and activation of AMPK in hepatocellular carcinoma.

doi: 10.3892/or.2013.2890

Figure Lengend Snippet: Figure 1. AZD8055-induced cell death is associated with caspase-dependent apoptotic signaling cascade. (A) AZD8055 induces cell death in a time- and concentration-dependent manner. Huh7 and Hep3B cells were treated with different concentrations (5, 10, 20, 50 and 100 nM) of AZD8055 for 24 h or were treated with 100 nM AZD8055 for variable periods (6, 12, 24 and 48 h). Cell death was assessed by performing MTT assay and trypan blue staining, respectively. Data represent the mean ± SD derived from at least 3 separate experiments. *P<0.05, **P<0.01, compared to control. (B) AZD8055 suppresses colony formation of HCC cells. Huh7 and Hep3B cells were plated in 6-well plates in RPMI-1640 media containing 100 nM AZD8055 and colonies were stained with crystal violet and counted in triplicate wells after growth for a further 2 to 3 weeks. DMSO was used as control. Data represent the mean ± SD derived from at least 3 separate experiments. **P<0.01. (C) AZD8055 does not cause caspase activation. Huh7 and Hep3B cells were treated with 100 nM AZD8055 for 24 h and immunoblot analysis was performed to detect PARP cleavage and caspase-3, caspase-9 activation. β-actin was used as a loading control (up). Quantitative analysis of cas pase-3, caspase-8 and caspase-9 activity was assessed by colorimetric assay (down). Data represent the mean ± SD derived from at least 3 separate experiments.

Article Snippet: AZD8055 was purchased from Selleck Chemicals, dissolved in dimethyl sulfoxide (DMSO) as a stock concentration of 1 mmol/l and diluted to the indicated concentrations with culture medium.

Techniques: Concentration Assay, MTT Assay, Staining, Derivative Assay, Control, Activation Assay, Western Blot, Activity Assay, Colorimetric Assay

Journal: Oncology reports

Article Title: AZD8055 induces cell death associated with autophagy and activation of AMPK in hepatocellular carcinoma.

doi: 10.3892/or.2013.2890

Figure Lengend Snippet: Figure 2. AZD8055 induces autophagy in HCC cells. (A) Effect of AZD8055 on intracellular localization of LC3 protein. Huh7 cells were treated with 100 nM AZD8055 or with the same volume of DMSO as control for 12 h. Immunofluorescence staining was performed to examine the transformation of cytosolic LC3-I to membrane-bound LC3-II. Quantitatively, percentage of cells with dots of LC3 in control or AZD8055-treated cells were assessed by counting at least 100 cells under fluorescence microscopy (right). Each column represents mean ± SD of 3 independent experiments. **P<0.01. (B) Effect of AZD8055 on the expression of Atg-5/12, BECN1 and LC3-II in human HCC cell lines. Huh7 cells were treated with 100 nM AZD8055 for the indicated times. After treatment, cell extracts were prepared for immunoblot analysis. The levels of Atg-5/12, BECN1 and LC3-II were detected using the corresponding antibodies. β-actin was used as a loading control (left). The amount of Atg-5/12, BECN1 and LC3-II was quantified by a laser densitometer. After normalization to β-actin, the relative levels of Atg-5, BECN1 and LC3-II were compared with those at time 0 as a value of one. Data represent mean ± SD of 3 independent experiments (right).

Article Snippet: AZD8055 was purchased from Selleck Chemicals, dissolved in dimethyl sulfoxide (DMSO) as a stock concentration of 1 mmol/l and diluted to the indicated concentrations with culture medium.

Techniques: Control, Immunofluorescence, Staining, Transformation Assay, Membrane, Fluorescence, Microscopy, Expressing, Western Blot

Journal: Oncology reports

Article Title: AZD8055 induces cell death associated with autophagy and activation of AMPK in hepatocellular carcinoma.

doi: 10.3892/or.2013.2890

Figure Lengend Snippet: Figure 3. Effects of 3-MA on AZD8055-induced cell death in HCC cells. (A) 3-MA decreases LC3-II expression in Huh7 and Hep3B cells. Cells were treated with DMSO or 100 nM AZD8055 for 12 h following 2-h pretreatment of 2 mM 3-MA. After treatment, cell extracts were prepared for immunoblot analysis. The levels of LC3-II were detected using the corresponding antibodies. β-actin was used as a loading control (left). After normalization to β-actin, the relative levels of LC3-II were compared with those at time 0 as a value of one. Data represent mean ± SD of 3 independent experiments (right). (B) Inhibition of autophagy by 3-MA decreases cell death in Huh7 and Hep3B cells. Cells were treated with DMSO or 100 nM AZD8055 for 12 h following 2-h pretreatment of 2 mM 3-MA. Cell death was assessed by trypan blue staining. Data represent the mean ± SD derived from at least 3 separate experiments. *P<0.05, **P<0.01.

Article Snippet: AZD8055 was purchased from Selleck Chemicals, dissolved in dimethyl sulfoxide (DMSO) as a stock concentration of 1 mmol/l and diluted to the indicated concentrations with culture medium.

Techniques: Expressing, Western Blot, Control, Inhibition, Staining, Derivative Assay

Journal: Oncology reports

Article Title: AZD8055 induces cell death associated with autophagy and activation of AMPK in hepatocellular carcinoma.

doi: 10.3892/or.2013.2890

Figure Lengend Snippet: Figure 4. Role of AMPK activation in AZD8055-induced cell death. (A) Cells were treated with 100 nM AZD8055 for the indicated times. After treatment, cells were harvested and cell extracts were prepared for immunoblot analysis. The levels of AMPK and p-AMPK were detected by immunoblot analysis using the corresponding antibodies. β-actin was used as a loading control. The amount of AMPK and p-AMPK was quantified by a laser densitometer. After normalization to β-actin, the relative levels of AMPK and p-AMPK were compared with those at time 0 as a value of one. Data represent mean ± SD of 3 independent experiments (right). *P<0.05, **P<0.01. (B) Blocking AMPK activation by dorsomorphin increases cell death in Huh7 and Hep3B cells. Cells were treated with DMSO or 100 nM AZD8055 for 12 h following 2-h pretreatment of 2 mM dorsomorphin. Cell death was assessed by trypan blue staining. Data represent the mean ± SD derived from at least 3 separate experiments. (C) Blocking AMPK activation by dorsomorphin decreases LC3-II expression in Huh7 and Hep3B cells. Cells were treated with DMSO or 100 nM AZD8055 for 12 h following 2-h pretreatment of 2 mM dorsomorphin. After treatment, cell extracts were prepared for immunoblot analysis. The levels of LC3-II were detected using the corresponding antibodies. β-actin was used as a loading control (left). Data represent means ± SD of 3 independent experiments (right). *P<0.05, **P<0.01.

Article Snippet: AZD8055 was purchased from Selleck Chemicals, dissolved in dimethyl sulfoxide (DMSO) as a stock concentration of 1 mmol/l and diluted to the indicated concentrations with culture medium.

Techniques: Activation Assay, Western Blot, Control, Blocking Assay, Staining, Derivative Assay, Expressing