Fig 1.
The molecular structure of DR and its cytotoxic effects on various cancer cell lines.
(A) The molecular structures of the DR. Non-small cell lung cancer A549 cells (B), mucoepidermoid pulmonary cancer H292 cells (C), prostate cancer PC3 cells (D), and colon cancer HCT116 cells (E) were treated with the DR of indicated concentrations for 24 h or 48 h, and then measured by MTT assay. All data are expressed as the mean ± SD obtained from at least three independent experiments. Differences were considered significant at p<0.05 (*), p< 0.01 (**), and p< 0.001 (***) compared with the DMSO control.
Fig 2.
DR induces non-apoptotic cell death at low concentrations.
(A) DR was treated with various concentration for 24 h, and cell cycle analysis was performed using flow cytometry. (B) The cell cycle distribution was quantified and represented as a bar graph. (C) After 24 h incubation with the indicated concentrations of DR, the A549 cells were lysed and aliquots were assayed for in vitro caspase-8, - 9 and -3 activity. (D) After DR treatment for 24 h, the cells were stained with Annexin V. Early apoptotic Annexin V-positive cells were detected by flow cytometry. (E) After treatment of DR with the indicated concentrations, the cells were lysated and analyzed by western blotting. (F) Cells were co-treated with pan caspase inhibitor (Z-VAD-fmk, 20 μM) and cell viability were measured by MTT assay. Statistical differences were presented p<0.05 (*), p<0.01 (**), and p<0.001 (***) compared with the DR alone; p<0.01 (##) compared with the DMSO control.
Fig 3.
DR induced autophagy in A549 cells.
(A) After A549 cells were treated with DR, the morphological change of cells was observed under the microscope. (B) Cells were treated with 60 μM DR for 6 h and observed under transmission electron microscopy. Arrowheads indicate autophagosome and arrows denote the vacuoles. (C) Cells were treated various concentrations of DR for 24 h before the western blot analysis. (D) Cells were treated 40 μM DR for 24 h with or without 1 h pretreatment with 0.5 μM wortmannin (Wort) or 20 μM chloroquine (CQ) and the morphological change of cells was observed under the microscope. (E) Cell viability was measured by MTT assay. Statistical differences were presented p<0.05 (*) compared with the DR alone; p<0.01 (##) compared with the DMSO control. (F) Cells were treated DR for 24h and immunoblot analysis was performed after cell lysis.
Fig 4.
Increased ROS production by DR treatment is related to autophagy.
(A) After A549 cells were treated with DR, the cells were loaded with the hydrogen peroxide sensitive dye H2DCFDA or the superoxide sensitive dye HE at 37°C for 30 min, and ROS generation was then measured using flow cytometry. (B) Cells were treated 40 μM DR for 24 h with or without treatment with NAC, GSH, and MnTBAP. The cell viability was measured by WST assay. (C) The morphological change of cells was observed under the microscope. (D) The cells were incubated with DiOC6(3) for 30 min 37°C in the dark, and analyzed by a flow cytometery. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 40 nM) was used as a positive control. Statistical differences were presented p<0.05 (*), p<0.01 (**), and p<0.001 (***) compared with the DR alone; p<0.01 (##) compared with the DMSO control.
Fig 5.
DR increases cytoplasmic Ca2+ concentration.
(A) DR was added to 1 μg/ml Fluo-4 AM preloaded A549 cells and the changes of intracellular Ca2+ were examined by flow cytometry. Cells were stained with 1 μg/ml Fluo-4 AM for 30 min and treated 40 μM DR with or without co-treatment with 10 μM BAPTA and/or 10 μM BAPTA-AM for 1 min. (B) Cytoplasmic Ca2+ was quantified and represented as a bar graph. (C) The morphological change of cells was observed under the microscope. (D) Cell viability was measured using MTT assay. (E) The cells were stained with 40 nM DiOC6(3) for 30 min at 37°C, and then analyzed by flow cytometry. (F) Cells were treated 40 μM DR for 24 h with or without treatment with 2 μM cyclosporine A. The cell viability was measured by MTT assay. All data are expressed as the mean ± S.D. obtained from at least three independent experiments. Statistical differences were presented p<0.01 (**) and p<0.001 (***) compared with the DR alone; p<0.01 (##) and p<0.001 (###) compared with the DMSO control.
Fig 6.
DR induces autophagy through ERK-mediated pathway.
(A) After treatment of 40 μM DR for the indicated times, the A549 cells were lysated and detected p-ERK by western blotting. (B) Cells were pretreated with U0126 (ERK inhibitor) further treated with DR for 24 h. Western blotting of the indicated proteins was performed, with tubulin detected as a loading control. (C) Cell viability was analyzed MTT assay. All data are expressed as the mean ± S.D. obtained from at least three independent experiments. Statistical differences were considered significant if p<0.05 (*), p<0.01 (**), and p<0.001 (***) compared with the DR alone; p<0.01 (##) compared with the DMSO control.