Pegylated Recombinant Human Arginase 1 Induces Autophagy and Apoptosis via the ROS-Activated AKT/mTOR Pathway in Bladder Cancer Cells

Figure 5

BCT-100-induced ROS initiated cellular apoptosis and autophagy in bladder cancer cells. (a) ROS levels determined by flow cytometry in T24 cells pretreated with NAC (5 mM, 1 h) before incubation with BCT-100 (20 mU/ml) for 24 h and H2DCFDA (1 μM). (b) Cell apoptotic rate determined by flow cytometry of Annexin V/PI staining in T24 cells pretreated with NAC (5 mM, 1 h) before incubation with BCT-100 (20 mU/ml) for 72 h. (c) Expression of AKT, p-AKT, C-PARP, p62, LC3B, and β-actin evaluated by Western blot. (d) Expression of C-PARP, Survivin, LC3B, and β-actin evaluated by Western blot in T24 cells treated with BCT-100 (20 mU/ml) and CQ (10 μM) for 3 days. (e) Autophagic flux assessed by confocal microscopy in mRFP-GFP-LC3-transfected T24 cells treated with BCT-100 (20 mU/ml) and CQ (10 μM) for 3 days. (f, g) Expression levels of related proteins tested by Western blot in cells treated with MK-2206 (2 μM) and rapamycin (100 nM) with or without BCT-100 for 3 days. β-Actin was used as a loading control. All data are shown as the of three independent assays. versus control and ^# versus BCT-100, as evaluated by Student’s -test.

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