QBC939 cells are resistant to cisplatin.

<p>QBC939 and HepG2 cells were treated with cisplatin for 12 or 24 h, and then cell viability, apoptosis and cell death rates, and mtROS were measured. (A) Cells were treated with cisplatin (1.25–80 μg/ml) for 24 h and then cell viability was determined by MTT assays. (B) Cells were treated with cisplatin (10 or 20 μg/ml) for 12 h and then the apoptotic rate was measured by flow cytometry (fluorescence intensity: x axis, Annexin V-FITC; y axis, PI). (C) Quantitation of the apoptosis rate (including early and late apoptosis) under the same treatment conditions as in (B). (D) Quantitation of the cell death rate by trypan blue staining under the same treatment conditions as in (B). (E) Cells were treated with cisplatin (20 μg/ml) for 24 h followed by 5 μm mitoSOX for 10 min, and then fluorescence intensity was observed by fluorescence microscopy (×200). (F) Quantitation of the cell average fluorescence intensity under the same treatment conditions as in (E). All values are the mean±SE. *<i>p</i><0.05 between QBC939 cells group and HepG2 cells group.</p

CQ inhibited autophagy and enhanced ROS in QBC939 cells.

<p>(A) Cells were treated with CQ (50 μM) or 3-MA (5 mM) and/or cisplatin (10 or 20 μg/ml) for 24 h and then cell viability was measured by MTT assays. (B) Immunoblot analysis of intracellular p62 and LC3-II/I in cells treated with CQ (50 μM) or 3-MA (5 mM) for 24 h. Overall ROS and mtROS were measured in (C) QBC939 cells and HepG2 cells treated with 100 μM Mito-TEMPO and/or CQ (50 μM) or 3-MA (5mM) for 12 h (×200). Quantitation of the cell average fluorescence intensity of (D) DCFH-DA and (E) MitoSOX under the same treatment conditions as in (C). All values are the mean±SE.</p

Inhibition of glucose metabolism reduces the antioxidant capacity of QBC939 cells.

<p>Cells were treated with 20 mM 2-DG and 500 μM DHEA for 24 h and assayed for changes in the level of overall ROS and mtROS in (A) QBC939 cells and (B) HepG2 cells (×200). Quantitation of the cell average fluorescence intensity of (C) DCFH-DA and (D) MitoSOX under the same treatment conditions as in (A) and (B). Cells were treated with (E) 10 mM 2-DG or (F) 500 μM DHEA, alone or with the combination of cisplatin (10 or 20 μg/ml) respectively, for 24 h and then analyzed by MTT assays to detect cell viability. All values are the mean±SE.</p

CQ reduced the glucose metabolism-related antioxidant capacity and mitochondrial membrane potential in QBC939 cells and increases the amount of intracellular hydroxyl free radicals.

<p>Cells were treated with CQ (50 μM) or 3-MA (5 mM) for 6 h and then assayed for (A) intracellular G6PDH activity, (B) the NADPH/NADP ratio, and (C) GSH/GSSG ratio. (D) Cells treated with CQ (50 μM) or 3-MA (5 mM) were incubated for 8 h and then assayed for the mitochondrial membrane potential with JC-1 by flow cytometry (fluorescence intensity: x axis, green; y axis, red). (E) Cells treated with CQ (50 μM) or 3-MA (5 mM) were incubated for 24 h and then assayed for the amount of intracellular hydroxyl free radicals. All values are the mean±SE. *<i>p</i><0.05 between control and other group in QBC939 cells.</p

CQ enhanced cisplatin-induced apoptosis in QBC939 cells.

<p>(A) QBC939 cells and (B) HepG2 cells were treated with CQ (50 μM) or 3-MA (5 mM) and/or cisplatin (20 μg/ml) and/or Mito-TEMPO (100 μM) for 12 h or 8 h and then assayed for the level of mtROS (×200) and apoptosis rate was measured by flow cytometry, respectively. Quantitation of apoptosis rate (including early and late apoptosis) was shown in (C). All values are the mean±SE. ^a,b<i>p</i><0.05 between with and without Mito-TEMPO group in HepG2 cells, ^c,d<i>p</i><0.05 between with and without Mito-TEMPO group in QBC939 cells.</p