Transient Alteration of Cellular Redox Buffering before Irradiation Triggers Apoptosis in Head and Neck Carcinoma Stem and Non-Stem Cells

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive and recurrent malignancy owing to intrinsic radioresistance and lack of induction of apoptosis. The major focus of this work was to design a transient glutathione depleting strategy during the course of irradiation of HNSCC in order to overcome their radioresistance associated with redox adaptation. Methodology/Principal Findings: Treatment of SQ20B cells with dimethylfumarate (DMF), a GSH-depleting agent, and L-Buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis 4 h before a 10 Gy irradiation led to the lowering of the endogenous GSH content to less than 10 % of that in control cells and to the triggering of radiation-induced apoptotic cell death. The sequence of biochemical events after GSH depletion and irradiation included ASK-1 followed by JNK activation which resulted in the triggering of the intrinsic apoptotic pathway through Bax translocation to mitochondria. Conclusions: This transient GSH depletion also triggered radiation-induced cell death in SQ20B stem cells, a key event to overcome locoregional recurrence of HNSCC. Finally, our in vivo data highlight the relevance for further clinical trials o

Central role of spatial ROS distribution at the nanometric scale in the molecular response to carbon ion irradiation

International audienceBackground: Hadrontherapy is an alternative to radiotherapy in the treatment of Head-and-Neck cancers (HNSCC), because of accurate ballistic and high biological efficiency, even in hypoxic tumor areas. These cancers are of poor prognosis because of a high risk of recurrence related to the presence of cancer stem cells (CSCs) located in hypoxic niches.Aim of this work was to determine the molecular specificities of the response to carbon-ion irradiation versus photons in HNSCC cancer cell lines and their CSCs’ subpopulation, under hypoxic and normoxic conditions.Methods : SQ20B, FaDu cells, and their CSCs were irradiated with photons or carbon-ion (290MeV/n,NIRS) in normoxia or hypoxia (1%O2). Cell survival curves, expression of HIF-1a reactive oxygen species (ROS) and Migration/Invasion processes were quantified.Results/Conclusions: For CSCs and non-CSCs, an oxygen-enhancement-ratio (OER) upper than 1.2 was measured in response to photons, associated with stabilization of HIF-1α. This stabilization, depending on the ROS production, appears earlier in CSCs. Inhibition of HIF-1α expression results in decreased survival in HNSCC-CSCs after both type of radiation under hypoxia associated with a significant increase in residual DNA-DSBs. Furthermore, a relationship is demonstrated between HIF-1α expression and the DSBs’ detection and repair by the Homologous-Recombination. Finally, the dense and homogeneous ROS production induced by photons, essential for HIF-1α stabilization, leads to the activation of the 3 major epithelio-mesenchymal transition (EMT) signaling pathways (STAT3,MEK/p38/JNK,Akt/mTOR). At the opposite, the ROS concentrated into the carbon ion tracks are insufficient to activate HIF-1α and the upstream EMT pathways.All these results, supported by Monte-Carlo simulations, converge towards the central role of spatial ROS distribution at the nanometric scale to explain the specificities of the molecular response to carbon ions. Their therapeutic advantage may result both from unrepaired complex-DNA lesions and the non-activation of ROS-dependent-signaling pathways involved in tumor cell defense.Supported by Labex PRIMES-ANR-11-LABX-0063;ANR-11-IDEX-0007;ITMO-Cancer-AVIESA

Differential Formation of Stress Granules in Radiosensitive and Radioresistant Head and Neck Squamous Cell Carcinoma Cells

International audiencePurpose: Stress granules (SGs) are cytoplasmic aggregates in which mRNAs and specific proteins are trapped in response to a variety of damaging agents. They participate in the cellular defense mechanisms. Currently, their mechanism of formation in response to ionizing radiation and their role in tumor-cell radiosensitivity remain elusive.Methods and materials: The kinetics of SG formation was investigated after the delivery of photon irradiation at different doses to head and neck squamous cell carcinoma cell lines with different radiosensitivities and the HeLa cervical cancer cell line (used as reference). In parallel, the response to a canonical inducer of SGs, sodium arsenite, was also studied. Immunolabeling of SG-specific proteins and mRNA fluorescence in situ hybridization enabled SG detection and quantification. Furthermore, a ribopuromycylation assay was used to assess the cell translational status. To determine whether reactive oxygen species were involved in SG formation, their scavenging or production was induced by pharmacologic pretreatment in both SCC61 and SQ20B cells.Results: Photon irradiation at different doses led to the formation of cytoplasmic foci that were positive for different SG markers. The presence of SGs gradually increased from 30 minutes to 2 hours postexposure in HeLa, SCC61, and Cal60 radiosensitive cells. In turn, the SQ20B and FaDu radioresistant cells did not form SGs. These results indicated a correlation between sensitivity to photon irradiation and SG formation. Moreover, SG formation was significantly reduced by reactive oxygen species scavenging using dimethyl sulfoxide in SCC61 cells, which supported their role in SG formation. However, a reciprocal experiment in SQ20B cells that depleted glutathione using buthionine sulfoximide did not restore SG formation in these cells.Conclusions: SGs are formed in response to irradiation in radiosensitive, but not in radioresistant, head and neck squamous cell carcinoma cells. Interestingly, compared with sodium arsenite-induced SGs, photon-induced SGs exhibited a different morphology and cellular localization. Moreover, photon-induced SGs were not associated with the inhibition of translation; rather, they depended on oxidative stress

Circulating Tumor Cell Detection during Neoadjuvant Chemotherapy to Predict Early Response in Locally Advanced Oropharyngeal Cancers: A Prospective Pilot Study

Patients with locally advanced oropharyngeal carcinoma treated with neoadjuvant chemotherapy are reassessed both radiologically and clinically to adapt their treatment after the first cycle. However, some responders show early tumor progression after adjuvant radiotherapy. This cohort study evaluated circulating tumor cells (CTCs) from a population of locally advanced oropharyngeal carcinoma patients treated with docetaxel, cisplatin, and 5-fluorouracil (DCF) induction chemotherapy or DCF with a modified dose and fractioned administration. The counts and phenotypes of CTCs were assessed at baseline and at day 21 of treatment, after isolation using the RosetteSepTM technique based on negative enrichment. At baseline, 6 out of 21 patients had CTCs (28.6%). On day 21, 5 out of 11 patients had CTCs (41.6%). There was no significant difference in the overall and progression-free survival between patients with or without CTCs at baseline (p = 0.44 and 0.78) or day 21 (p = 0.88 and 0.5). Out of the 11 patients tested at day 21, 4 had a positive variation of CTCs (33%). Patients with a positive variation of CTCs display a lower overall survival. Our findings suggest that the variation in the number of CTCs would be a better guide to the management of treatment, with possible early changes in treatment strategy

<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

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

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

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

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

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