Reduction in c-FLIP_L level following hyperthermia and oxaliplatin in CX-1 cells.

<p>(A) Cells were exposed to 37°C or 42°C for 1 h in the presence/absence of 10 µg/ml oxaliplatin and then harvested immediately or 3 h after incubation at 37°C. c-FLIP_L was examined by Western blot analysis. (B) Cells were exposed to 37°C or 42°C for 1 h in the presence/absence of 10 µg/ml oxaliplatin and then incubated for 3 h at 37°C. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to measure relative c-FLIP mRNA level. The bar graph represents mean values (+SD) from triplicate experiments. (C) Cells were exposed to 37°C or 42°C for 1 h in the presence/absence of 10 µg/ml oxaliplatin and then incubated for 3 h at 37°C. Protein synthesis was measured by [³⁵S] Methionine incorporation. (D) Cells were treated with 30 µg/ml CHX, or exposed to hyperthermia in the presence or absence of CHX. The levels of c-FLIP_L and loading control actin were measured by Western blot analysis. (E) Cells were exposed to hyperthermia for 30 or 60 min in the presence/absence of MG132. Lysate samples were immunoprecipitated with anti-ubiquitin antibody and protein G-Sepharose. The ubiquitinated FLIP was detected by Western blot with anti-FLIP antibody. (F) Cells were transiently transfected with 4 µg plasmid containing mock, K106R (106 lysine residue was replaced with arginine), K195R, or wild-type (WT) c-FLIP_L. After 48 h incubation, cells were exposed to hyperthermia at 42°C for 1 h. The level of c-FLIP_L was detected by anti-FLIP antibody. Actin was used as an internal control. (G) Cells were transiently transfected with Flag-tagged c-FLIP_L WT or K195R plasmid; 48 h later, cells were subjected to hyperthermia at 42°C for 1 h. The levels of ubiquitinated c-FLIP_L were detected by IP with anti-Flag antibody followed by Western blot using anti-ubiquitin antibody. The presence of transfected c-FLIP_L in the lysates was verified by Western blot. Actin was shown as an internal standard. (H) Cells were transiently transfected with c-FLIP_L WT, K106R, or K195R plasmid; 48 h later, cells were heated at 42°C for 1 h in the presence/absence of Mapa (100 ng/ml) and oxaliplatin (10 µg/ml) and then incubated at 37^°C for 3 h. Lysates containing equal amounts of protein were immunoblotted with anti-PARP and anti-FLIP antibody. Actin was shown as an internal standard. (I) Cells were transiently transfected with c-FLIP_L WT, K106R, or K195R plasmid; 48 h later, cells were heated at 42°C for 1 h in the absence or presence of Mapa (100 ng/ml) and oxaliplatin (10 µg/ml), and then incubated for 24 h at 37°C. Cell viability was analyzed by MTS assay. Error bars represent SD from triplicate experiments. Asterisk * represents a statistically significant difference (P <0.05).</p

Effect of oxaliplatin and hyperthermia on Mapa-induced cytotoxicity and apoptosis.

<p>(A, B) CX-1 and HCT116 cells were exposed to normothermic or hyperthermic (42°C) conditions for 1 h in the presence/absence of Mapa and oxaliplatin and then incubated for 23 h at 37°C in the presence/absence of Mapa and oxaliplatin. Cell viability was analyzed by MTS assay. Error bars represent SD from triplicate experiments. Asterisk ** represents a statistically significant difference (P <0.01). (C) CX-1 cells were exposed to hyperthermia (42°C) for 1 h in the presence/absence of Mapa and oxaliplatin and then incubated for 3 h at 37°C in the presence/absence of Mapa and oxaliplatin. After treatment, cells were stained with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI). Apoptosis was detected by the flow cytometric assay. (D) After treatment, the cleavage of caspase 8, caspase 9, caspase 3, or PARP was detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in each lane. (E) CX-1 and HCT116 cells were treated with or without 20 µM Z-IETD-FMK (caspase 8 inhibitor), Z-LEHD-FMK (caspase 9 inhibitor), and Z-DEVD-FMK (caspase 3 inhibitor) for µmin followed by oxaliplatin/Mapa/hyperthermia and the cleavage of PARP was detected by immunoblotting. (F) Human colon cancer stem cells, Tu-12, Tu-21 and Tu-22, were exposed to normothermic or hyperthermic (42°C) conditions for 1 h in the presence/absence of Mapa and oxaliplatin at the indicated concentration and then incubated for 23 h at 37°C in the presence/absence of Mapa and oxaliplatin. PARP was detected by immunoblotting. Actin was used as loading control.</p

Multimodality treatment-induced JNK phosphorylation, Bcl-xL phosphorylation and reduction in c-FLIP_L level.

<p>(A) CX-1 and (B) HCT116 cells were exposed to hyperthermia (42°C) for 1 h in the presence/absence of Mapa and oxaliplatin and incubated for 3 h at 37°C in the presence/absence of Mapa and oxaliplatin. After treatment, the cleavage of caspase 8, caspase 9, caspase 3, or PARP was detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in each lane. (C) CX-1 cells were exposed to hyperthermia (42°C) for 1 h in the presence/absence of 100 ng/ml Mapa and 10 µg/ml oxaliplatin and then incubated for 3 h at 37°C in the presence/absence of Mapa and oxaliplatin. After treatment, cells were immunoblotted with anti-phospho-JNK/JNK, anti-phospho-Bcl-xL/Bcl-xL and anti-FLIP antibodies. (D) CX-1 cells were exposed to hyperthermia (42°C) for 1 h in the presence/absence of Mapa (100 ng/ml-1000 ng/ml) and oxaliplatin (10 µg/ml-100 µg/ml) and incubated for 3 h at 37°C in the presence/absence of Mapa and oxaliplatin. After treatment, phospho-JNK/JNK, phospho-Bcl-xL/Bcl-xL and FLIP_L were detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in each lane. (E) HCT116 cells were exposed to hyperthermia (42°C) for 1 h in the presence/absence of Mapa (10 ng/ml-100 ng/ml) and oxaliplatin (10 µg/ml-100 µg/ml) and then incubated for 3 h at 37°C in the presence/absence of Mapa and oxaliplatin. After treatment, phospho-JNK/JNK, phospho-Bcl-xL/Bcl-xL and FLIP_L were detected by immunoblotting. Actin was used to confirm the equal amount of proteins loaded in each lane.</p

The kinetics of multimodality treatment in CX-1 and HCT116 cells.

<p>CX-1 (A) and HCT116 (B) cells were exposed to hyperthermic (42°C) conditions for 1 h in the presence/absence of Mapa and oxaliplatin and incubated at 37°C in the presence/absence of Mapa and oxaliplatin for 3 h, 7 h, 11 h and 23 h. After treatment, the cleavage of caspase 8/9/3 and PARP, phospho-JNK/JNK, phospho-Bcl-xL/Bcl-xL and FLIP_L were detected by immunoblotting. Actin was used to confirm the equal amount of proteins.</p

Protection from multimodality treatment-induced apoptosis by overexpression of c-FLIP_L WT/K195R and Bcl-xL-S62A in CX-1 or HCT116 cells.

<p>(A) CX-1 cells stably overexpressed with pcDNA, c-FLIP_L WT, Bcl-xL-S62A and c-FLIP_L WT + Bcl-xL-S62A, and three stable clones were pooled, and cells were heated at 42°C for 1 h in the presence/absence of Mapa (10 ng/ml) and oxaliplatin (10 µg/ml) and then incubated at 37^°C for 3 h. The cleavage of PARP, and the level of c-FLIP_L and Bcl-xL were detected by Western blot analysis. (B) Cell viability was analyzed by MTS assay 24 h after treatment. Error bars represent SD from triplicate experiments. Asterisk ** represents a statistically significant difference (P <0.01). (C) HCT116 cells were transiently transfected with equal amount of plasmid containing mock, c-FLIP_L K195R, Bcl-xL-S62A and c-FLIP_L K195R + Bcl-xL-S62A. After 48 h incubation, cells were heated at 42°C for 1 h in the presence/absence of Mapa (10 ng/ml) and oxaliplatin (10 µg/ml) and then incubated at 37^°C for 3 h. The cleavage of PARP, and the level of c-FLIP_L and Bcl-xL were detected by Western blot analysis. (D) Cell viability was analyzed by MTS assay 24 h after treatment. Error bars represent SD from triplicate experiments. Asterisk * represents a statistically significant difference (P <0.05).</p

The requirement of phosphorylation of JNK and Bcl-xL in the multimodality treatment-induced apoptosis in CX-1 cells.

<p>(A) Cells were pretreated with JNK inhibitor 25 µM SP6001125 followed by oxaliplatin/Mapa/hyperthermia and immunoblotted with anti-PARP, anti-phospho-Bcl-xL and anti-Bcl-xL antibody. (B) Transfectants with control plasmid (pcDNA), wild-type Bcl-xL (Bcl-xL-WT), Ser62/Ala phospho-defective Bcl-xL mutant (Bcl-xL-S62A), or Ser62/Asp phospho-mimic Bcl-xL mutant (Bcl-xL-S62D) were treated with oxaliplatin/Mapa/hyperthermia and immunoblotted with anti-PARP or anti-Bcl-xL antibody. Actin was used to confirm the equal amount of proteins loaded in each lane.</p

Role of anti-oxidant agents in radiosensitivity of non-stem cells and CSC-like cells.

<p>(<b>A</b>) Non-stem (N) and stem-like (S) MDA-MB-453 and MDA-MB-231 cells were harvested. Lysates containing equal amounts of protein (20 µg/ml) were separated by SDS-PAGE, and immunoblotted with anti-MnSOD, anti-CuZnSOD, or anti-catalase antibody. Actin was shown as an internal standard. (<b>B</b>) Densitometry analysis of each band was performed. The area integration of optical density of each band in stem-like cells (S) was compared with that in non-stem cells (NS). Error bars represent standard error from the mean for three separate experiments. (<b>C</b>) MDA-MB-453 non-stem cells and stem-like cells were treated with 200 µM L-buthionine-sulfoximine (BSO) for 24 hr and GSH content was determined. Error bars represent standard error from the mean for three separate experiments. (<b>D</b>) BSO-treated/untreated non-stem and stem-like MDA-MB-453 and MDA-MB-231 cells were irradiated at 6.25 Gy and survival was determined. Error bars represent standard error from the mean for three separate experiments.</p

Percentage of cells stained γ-H2AX positive after irradiation at 2.5 Gy.

<p>Kinetics of γ-H2AX foci removal after irradiation. Non-stem and CSC-like MDA-MB-435 and MDA-MB-231 cells were irradiated at 2.5 Gy. Various times (0.5–12 hr) after irradiation, cells were fixed and immunostained with anti-phospho-H2AX antibody. Nuclei containing at least six fluorescent foci were considered positive and percentage of cells stained γ-H2AX positive was determined. Error bars represent standard error from the mean for three separate experiments.</p

Characterization of breast cancer stem cell-like (CSC-like) cells.

<p>(<b>A</b>) Parental MDA-MB-453 cells were transfected with a plasmid encoding GFP under the control of the CMV promoter (non-stem) or Oct3/4 promoter (stem-like). After selection in G-418, GFP+ colonies were pooled. Phase-contrast images and fluorescence images of parental cells (Parent: a, d), CMV-GFP-transfected non-stem cells (Non-stem: b, e) or Oct3/4-GFP-transfected CSC-like cells (Stem-like: c, f) were visualized by light (<b>Phase-contrast: a–c</b>) or UV (<b>Fluorescence: d–f</b>) microscopy. (<b>B</b>) Flow cytometry characterization of parental, non-stem, or CSC-like cells was performed. CMV promoter-driven GFP cDNA (<b>e;</b> non-stem cells) or human Oct3/4 promoter-driven GFP cDNA (<b>f;</b> stem-like cells) transfected MDA-MB-453 cells were stained with surface marker antibodies (CD24, CD44) and evaluated by flow cytometry. (<b>a–c</b>) Unstained cells and (<b>a, d</b>) parental cells. (<b>C</b>) Stem cell-associated Oct-4 gene expression was examined in MDA-MB-453 and MDA-MB-231 parental (P), non-stem (N) and CSC-cell like (S) cells. Cells were harvested with lysis buffer. Lysates containing equal amounts of protein (20 µg/ml) were separated by SDS-PAGE, and immunoblotted with anti-Oct-4 antibody. Actin was shown as an internal standard. (<b>D</b>) Mammosphere formation was compared in MDA-MB-231 and MDA-MB-453 CSC-like and non-stem cells. For mammosphere formation, 1,000 cells from stem-like cells or non-stem cells were plated into ultra-low attachment plates. Phase-contrast images of mammospheres of non-stem (left panels) or CSC-like (right panels) cells were obtained 4 days or 9 days later. (<b>E</b>) Xenograft tumor formation was established with CSC-like and non-stem MDA-MB-231 cells. For tumor formation in NOD/SCID mice, 1×10⁴ stem-like or non-stem cells were injected into the upper mammary fat pad. Tumor volumes were measured 30 days after injection.</p

Alkaline comet images and their quantitative analysis for non-stem and stem-like MDA-MB-231 cells after irradiation.

<p>(<b>A</b>) Control (0 Gy) or irradiated (6.25 Gy) cells for both non-stem cells (upper row) and stem-like cells (bottom row) were subjected to alkaline comet assay (refer to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050423#s2" target="_blank">Methods</a>) and the DNA was visualized by using a propidium iodide. (<b>B</b>) Distribution of comet tail moments for different treatments was plotted. Tail moments are the products of the distance of DNA migration (microns) and the amount of separated DNA (%).</p