Involvement of the MAPK pathway in the triggering of apoptosis after transient intracellular GSH depletion before irradiation.

<p>SQ20B cells were treated with 100 µM DMF and 100 µM BSO for 4 h whereas 10 µM SP600125, a specific JNK inhibitor, was added 1 h before irradiation in the cell culture medium before irradiation. The drugs were then removed by washing with fresh medium. After different points in time after irradiation, cells were harvested and the extracted proteins submitted to Western blot analysis. Panel A: Western blot analysis of phosphorylated Erk, p38 MAPK, and GAPDH. Panel B: Western blot analysis of inhibition of JNK phosphorylation by SP600125. Panel C: Western blot analysis of phosphorylated JNK and GAPDH. Panel D shows the consequence of JNK inhibition in terms of apoptosis estimated by flow cytometry through the total caspase activity (left) and the % of cells in sub-G1 phase (right) measurement 72 h after irradiation. Results are expressed as mean ± S.D. for three different experiments. The statistical significance is expressed as ***, p<0.001 <i>versus</i> 10 Gy only.</p

Combined treatment of DMF + BSO with irradiation enhances the survival of mice and inhibits tumor growth without apparent cytoxicity.

<p>Panel A shows the efficiency of a single intratumoural injection of 32 mg/kg DMF and 8 mg/kg BSO on the depletion of GSH within the tumour. Panel B shows the relative development of tumour size after combined DMF + BSO treatment each day whether or not associated with an irradiation dose of 20 Gy (4 Gy×5 days). The statistical significance is expressed as *, p<0.05, **, p<0.01 between treated and irradiated tumours versus irradiated tumours. Panel C shows the body weight monitoring of mice after the combined DMF + BSO treatment whether or not associated with an irradiation dose of 20 Gy (4 Gy×5 days). Panel D shows the Kaplan-Meyer survival curves representing the percentage of mice alive at the indicated points in time for each group of the experiment. Panel E shows the detection of apoptosis by TUNEL staining on paraffin-embedded tumor sections.</p

Activation of Ask1 upstream of the MAPK pathway in irradiated GSH-depleted SQ20B cells.

<p>SQ20B cells were treated with 100 µM DMF and 100 µM BSO for 4 h before irradiation. The drugs were then removed by washing with fresh medium. At different times post irradiation, cells were harvested and the extracted proteins submitted to Western blot analysis. Panel A: levels of phosphorylated Ask1 Panel B: levels of ASK-1 after specific siRNA transfection and downstream phosphorylation of JNK. Panel C: control of apoptosis through the measurement of total caspase activity 72 h after transfection of irradiated GSH-depleted cells with Ask1 SiRNA. Results are expressed as mean ± S.D. for three different experiments. The statistical significance is expressed as <b>***</b>, p<0.001 <i>versus</i> 10 Gy only.</p

Depletion of endogenous glutathione content before γ-ray exposure triggers radiation-induced intrinsic apoptosis in SQ20B cell line.

<p>SQ20B cells were treated with 100 µM DMF and 100 µM BSO for 4 h before irradiation. The drugs were then removed by washing with fresh medium. Total caspase activity and the percentage of cells in the sub-G1 phase were determined by flow cytometry, respectively after VAD-FMK-FITC (A) and propidium iodide (B) staining. Panel C shows the nuclear morphology of cells by DAPI staining 72 h post irradiation. Panels D and E show the mitochondrial alteration after JC-1 staining through the measurement by flow cytometry of the transmembrane potential (A) and after hydro-ethidine staining to measure the reactive oxygen species generated by respiratory chain (B). Results are expressed as mean ± S.D. for three different experiments in triplicate. The statistical significance is expressed as **, p<0.01 and ***, p<0.001 <i>versus</i> 10 Gy only.</p

Increase of intrinsic apoptosis in irradiated HNSCC cancer stem cells.

<p>After cell sorting, two sub-populations were obtained from the SQ20B carcinoma cell line: CD44⁺ cancer stems cells (A, C, E) and a CD44⁻ side population (B, D, F) which were grown in the SQ20B culture medium for 10 days. Both cell populations were treated with 100 µM DMF and 100 µM BSO for 4 h before irradiation and drugs were then removed by washing with fresh medium. At different times after irradiation, the percentage of apoptotic cells was quantified by flow cytometry analysis. Panels A and B: quantification of apoptotic cells in the sub-G1 phase after propidium iodide staining of CD44⁺ (A) and CD44⁻ (B) sub-populations. Panels C and D: quantification of ROS after hydro-ethidium staining of CD44⁺ (C) and CD44⁻ (D) sub-populations. Panels E and F: Quantification of the mitochondrial transmembrane potential decrease after JC-1 staining of CD44⁺ (E) and CD44⁻ (F) subpopulations. Results are expressed as mean ± S.D. for three different experiments. The statistical significance is expressed as <b>*</b>, p<0.05, <b>**</b>, p<0.01 and <b>***</b>, p<0.001 <i>versus</i> 10 Gy only.</p

Translocation of the pro-apoptotic protein Bax to mitochondria and release of cytochrome c in the cytosol of irradiated GSH-depleted SQ20B cells.

<p>SQ20B cells were treated with 100 µM DMF and 100 µM BSO for 4 h before irradiation. The drugs were then removed by washing with fresh medium. At different time post irradiation, mitochondria were isolated with standard fractionation procedure. The translocation of Bax to mitochondria (Panel A) and the release of cytochrome c in cytosol (Panel C) were measured by Western immunoblotting assay. In parallel, the activation of pro-caspase 8 and the cleavage of Bid were estimated by Western blot analysis (Panel B).</p

<i>In vitro</i> cytotoxicity and efficiency of the glutathione depleting strategy using the pharmacological association of DMF + BSO.

<p>SQ20B cells were plated and treated by DMF and/or BSO. Panel A shows the survival of SQ20B cells, determined by the MTT assay, after 24 h of continuous treatment with increasing concentrations of both drugs. Panel B shows the endogenous glutathione level, determined by HPLC, after continuous treatment with DMF (100 µM) and/or BSO (100 µM). In a second set of experiments, panel C shows the endogenous glutathione level with and without 10 Gy irradiation and after treatment with DMF (100 µM) and BSO (100 µM) for 4 h. The drugs were then removed by washing with fresh medium. Results are expressed as mean ± S.D. for three different experiments in triplicate.</p