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

DIC inhibits cell viability and induces apoptosis in SKOV3 cells.

<p><b>(A)</b> SKOV3 cells were treated with increasing concentrations of DIC for 24 h, and cell viability was measured by the MTT assay. <b>(B)</b> After the indicated treatments for 24 h, SKOV3 cells were stained with Hoechst 33342 and imaged by confocal microscopy (scale bar, 50 μm; arrows, Hoechst-positive apoptotic cells). <b>(C)</b> Cells were treated as indicated for 24 h, stained with annexin V and PI, and analyzed by flow cytometry. Representative flow cytometry images are shown on the left, and the quantification of annexin V⁺PI⁺ apoptotic cells is shown on the right. <b>(D)</b> Upon treatment, the protein levels of caspase-3, cleaved caspase-3, PARP, and cleaved PARP in SKOV3 cells were examined by western blot, with representative western blot images presented on the left and the quantitation of protein expression relative to β-actin (internal control) presented on the right. Data are presented as the mean ± SE from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, when compared to nontreated control cells.</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

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

DIC elevates the reactive oxygen species (ROS) level and disrupts the mitochondrial membrane potential (MMP).

<p>Upon treating SKOV3 or A2780 cells with 2% DMSO, 50 mM DCA, 100 μM DIC, or 200 μM DIC for 24 h, the ROS generation <b>(A and C)</b> and MMP level <b>(B and D)</b> were measured by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001, when compared to cells treated with DMSO.</p

DIC inhibits cell viability and induces apoptosis in A2780 cells.

<p><b>(A)</b> A2780 cells were treated with increasing concentrations of DIC for 24 h, and cell viability was measured by the MTT assay. <b>(B)</b> After the indicated treatments for 24 h, A2780 cells were stained with annexin V and PI, and analyzed by flow cytometry. <b>(C)</b> The quantification of annexin V⁺PI⁺ apoptotic cells. <b>(D)</b> Upon treatment, the protein levels of caspase-3, cleaved caspase-3, PARP, and cleaved PARP in A2780 cells were examined by western blot, with representative western blot images presented on the left and the quantitation of protein expression relative to β-actin (internal control) presented on the right. Data are presented as the mean ± SE from three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001, when compared to nontreated control 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

DIC suppresses tumor growth <i>in vivo</i>.

<p>SKOV3 xenografts were established in nude mice, and the mice were treated as indicated. <b>(A)</b> The body weights of mice from all groups were monitored during the 12-day treatment period. <b>(B)</b> The tumor volume and tumor weight were measured at the indicated time points during treatment and at the time of sacrifice, respectively. <b>(C)</b> The protein levels of PDK1, PDHA1, and p-PDHA1 were examined by western immunoblotting, with β-actin examined as the internal control. Representative western blot images are shown on the left, and the quantification of protein levels relative to β-actin are shown on the right. <b>(D)</b> The protein levels of caspase-3, cleaved caspase-3, PARP, and cleaved-PARP in the SKOV3 xenografts were examined by western immunoblotting. Representative western blot images are shown on the left, and the quantification of protein levels relative to β-actin are shown on the right. Data are presented as the mean ± SE for all mice in each group. *P < 0.05, **P < 0.01, ***P < 0.001, when compared to the control group. <b>(E)</b> Detection of apoptosis and p-PDHA1 by the TUNEL assay and immunohistochemistry, respectively, in xenograft samples from the indicated groups (scale bar, 100 μm; arrows, apoptotic cells).</p

DIC switches glucose metabolism from aerobic glycolysis to oxidative phosphorylation in ovarian cancer cells.

<p>SKOV3 or A2780 cells were treated with vehicle (DMSO), DCA (50 mM), DIC (100 μM), or DIC (200 μM) for 24 h. Extracellular glucose <b>(A and E)</b> and lactate <b>(B and F)</b> concentrations were determined in the culture media and expressed as a normalization to total protein amounts. The oxygen consumption rate (OCR) <b>(C and G)</b> and extracellular acidification rate (ECAR) <b>(D and H)</b> were measured after the treatment of ovarian cancer cells. Data are presented as the mean ± SE from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, when compared to cells treated with DMSO.</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