cytochrome-c-t and Oropharyngeal-Neoplasms

The aim of this study was to investigate how head and neck squamous cell carcinoma (HNSCC) tissue biopsies maintained in a pseudo in vivo environment within a bespoke microfluidic device respond to radiation treatment.. Feasibility study.. Tertiary referral center.. Thirty-five patients with HNSCC were recruited, and liver tissue from 5 Wistar rats was obtained. A microfluidic device was used to maintain the tissue biopsy samples in a viable state. Rat liver was used to optimize the methodology. HNSCC was obtained from patients with T1-T3 laryngeal or oropharyngeal SCC; N1-N2 metastatic cervical lymph nodes were also obtained. Irradiation consisted of single doses of between 2 Gy and 40 Gy and a fractionated course of 5×2 Gy. Cell death was assessed in the tissue effluent using the soluble markers lactate dehydrogenase (LDH) and cytochrome c and in the tissue by immunohistochemical detection of cleaved cytokeratin18 (M30 antibody).. A significant surge in LDH release was demonstrated in the rat liver after a single dose of 20 Gy; in HNSCC, it was seen after 40 Gy compared with the control. There was no significant difference in cytochrome c release after 5 Gy or 10 Gy. M30 demonstrated a dose-dependent increase in apoptotic index for a given increase in single-dose radiotherapy. There was a significant increase in apoptotic index between 1×2 Gy and 5×2 Gy.. M30 is a superior method compared with soluble markers in detecting low-dose radiation-induced cell death. This microfluidic technique can be used to assess radiation-induced cell death in HNSCC and therefore has the potential to be used to predict radiation response.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Death; Cytochromes c; Feasibility Studies; Head and Neck Neoplasms; Humans; Immunohistochemistry; Keratin-18; L-Lactate Dehydrogenase; Laryngeal Neoplasms; Liver; Microfluidic Analytical Techniques; Oropharyngeal Neoplasms; Radiotherapy Dosage; Rats; Rats, Wistar

We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Epidermal Growth Factor; Gelsolin; Gene Expression; Humans; Interferon-alpha; Oropharyngeal Neoplasms