epidermal-growth-factor and Oropharyngeal-Neoplasms

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

Eukaryotic translation initiation factor 5A (eIF-5A) is the only cell protein that contains the unusual basic amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed by the transfer of the butylamine portion from spermidine to the epsilon-amino group of a specific lysine residue of eIF-5A precursor and the subsequent hydroxylation at carbon 2 of the incoming 4-aminobutyl moiety. Agents that reduce cell hypusine levels inhibit the growth of mammalian cells. These observations suggest that hypusine is crucial for proliferation and transformation of eukaryotic cells. Here we have studied whether the inhibition of hypusine synthesis can potentiate the anti-cancer activity of the anti-tumour agents interferon-alpha (IFN alpha) and cytosine arabinoside (ara-C). We have found that IFN alpha increased epidermal growth factor receptor (EGF-R) expression, but reduced S phase and proliferative marker expression in human epidermoid KB cells and that this effect was antagonised by epidermal growth factor (EGF). Growth inhibition induced by IFN alpha was paralleled by decreased hypusine synthesis and, when EGF counteracted anti-proliferative effects, a reconstitution of hypusine levels was recorded. We also studied the effects of IFN alpha on the cytotoxicity of the recombinant toxin TP40 which inhibits elongation factor 2, another step of protein synthesis, through EGF-R binding and internalisation; IFN alpha induced an about 27-fold increase of TP40 cytotoxicity in KB cells. Ara-C, another antineoplastic agent commonly used in haematologic malignancies, induced both apoptosis and iron depletion in human acute myeloid leukaemic cells. The combination of ara-C and of the iron chelator desferioxamine, a strong inhibitor of hypusine synthesis, had a synergistic activity on apoptosis in these cells. The data strongly suggest that the post-translational modifications of eIF-5A could be a suitable target for the potentiation of the activity of anti-cancer agents.

Topics: Antineoplastic Agents; Cell Cycle; Cell Death; Chelating Agents; Cytarabine; Deferoxamine; Epidermal Growth Factor; ErbB Receptors; Eukaryotic Translation Initiation Factor 5A; Exotoxins; Humans; Interferon-gamma; Iron; Kinetics; Leukemia; Lysine; Oropharyngeal Neoplasms; Peptide Initiation Factors; Protein Processing, Post-Translational; RNA-Binding Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

We previously found that interferon alpha2 recombinant (IFNalpha) increases the expression of epidermal growth factor receptor (EGF-R) in the human epidermoid cancer KB cell line. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on KB cell cycle kinetics. IFNalpha (1000 i.u./ml) for 48 h decreased the S-phase fraction and diminished the expression of Ki67 and proliferating cell nuclear antigen on KB cells. Incubation of IFNalpha-treated KB cells with 10 nM EGF for 12 h reversed these effects. We then studied several biochemical markers of cell proliferation. Ornithine decarboxylase activity was decreased to about one-tenth by IFNalpha and partly restored by EGF. Hypusine is contained only in eukaryotic initiation factor 5A and its levels are correlated with cell proliferation. IFNalpha decreased hypusine synthesis by 75%; exposure of cells to EGF for 12 h restored hypusine synthesis almost completely. We also studied the effects of IFNalpha on the cytotoxicity of the recombinant toxin TP40, which inhibits elongation factor 2 through EGF-R binding and internalization. IFNalpha greatly enhanced the TP40-induced inhibition of protein synthesis in KB cells. In conclusion, IFNalpha, which affects protein synthesis machinery and increases EGF-R expression, enhances the tumoricidal activity of TP40 and hence could be useful in the setting of anti-cancer therapy.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Epidermal Growth Factor; Exotoxins; Humans; Interferon alpha-2; Interferon-alpha; Lysine; Ornithine Decarboxylase; Oropharyngeal Neoplasms; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

Previous studies have shown increasing evidence that TGF-alpha, a ligand for the often overexpressed EGF-receptor may be important for the oncogenesis and autocrine stimulation of the proliferation in head and neck cancers. The occurrence of TGF-alpha and its relation to the EGF-receptor still remain unclear. Twenty six specimens (primaries and metastasis) of oropharyngeal squamous cell carcinomas were investigated for the presence of transforming growth factor alpha (TGF-alpha), epidermal growth factor (EGF) and the EGF-receptor using a tissue extraction method and a "sandwich" immuno absorbent assay. In 77% of the specimens we found TGF-alpha, all had a significant amount of EGF-receptors, no EGF was found. No significant difference was noted for metastasis and primaries. No correlation was seen to the TNM stage and to the histological grading. There was an inverse statistical correlation between the TGF-alpha and the EGF-receptor concentration. High TGF-alpha concentrations were associated with low EGF-receptor concentration. Interestingly, even high TGF-alpha concentrations showed a lower limit of EGF-receptor concentrations which could not be passed. The present investigation gives a quantitative determination of the EGF-receptors and TGF-alpha in oropharyngeal carcinomas. The results indicate that a EGF-receptor/TGF-alpha complex could be functionally important for autocrine/paracrine stimulation.

Topics: Carcinoma; Epidermal Growth Factor; Humans; Oropharyngeal Neoplasms; Oropharynx; Transforming Growth Factor alpha