leupeptins and Tongue-Neoplasms

Cetuximab (Erbitux, C225) is a chimeric monoclonal antibody that binds to the extracellular domain of epidermal growth factor receptor (EGFR), inhibiting tumor growth, invasion, angiogenesis and metastasis. However, the mechanisms underlying the effect of Cetuximab in human oral squamous cell carcinoma (OSCC) remain unclear. Here, we report that Cetuximab modulates EGFR protein stability through the ubiquitin/proteasome pathway, resulting in the inhibition of human OSCC growth. Cetuximab significantly inhibited the migration and invasion of human OSCC cells by blocking epithelial/mesenchymal transition (EMT) and the AKT and ERK pathways. Furthermore, Cetuximab-inhibited cell growth by modulating the expression of integrin β5. Taken together, these results provide novel insights into the mechanism of Cetuximab action and suggest potential therapeutic strategies for OSCC.

Topics: Actins; Antibodies, Monoclonal, Humanized; Carcinoma, Squamous Cell; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cetuximab; Cysteine Proteinase Inhibitors; Epithelial-Mesenchymal Transition; ErbB Receptors; Humans; Integrin beta Chains; Leupeptins; MAP Kinase Signaling System; Neoplasm Invasiveness; Neovascularization, Pathologic; Oncogene Protein v-akt; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Tongue Neoplasms; Ubiquitin

Hypoxia-inducible factor-1 (HIF-1) is a tumor angiogenic transcription factor composed of an α and β subunit. We investigated the effect of glucosamine hydrochloride (GS-HCl) on the expression of HIF-1α and HIF-1β in serum‑treated YD-8 human tongue cancer cells. While long-term (24 h) treatment with GS-HCl strongly repressed the expression of HIF-1α and HIF-1β at both the protein and mRNA levels, short-term (4 h) GS-HCl treatment inhibited HIF-1α at the protein level. Short-term GS-HCl treatment also decreased phosphorylation of p70S6K and S6, translation-related proteins. However, the results of subsequent pharmacological inhibition and protein stability analyses indicated that HIF-1α protein downregulation induced by short-term GS-HCl treatment was not through modulation of the mTOR/p70S6K/S6 signaling pathways, the 26S proteasomal and lysosomal activities and HIF-1α protein stability. Importantly, our further analyses identified that HIF-1α protein downregulation induced by short-term GS-HCl treatment was blunted by exogenous administration of the citric acid cycle metabolites citrate and 2-oxoglutarate, but not the glycolytic end byproducts pyruvate and lactate. These findings demonstrate firstly that short-term GS treatment selectively downregulates HIF-1α at the protein level in YD-8 cells via interference of production of the citric acid cycle metabolites. It is proposed that short-term GS-HCl exposure may be applied for the treatment of oral tumors with high expression of HIF-1α.

Topics: Aryl Hydrocarbon Receptor Nuclear Translocator; Cell Line, Tumor; Citric Acid; Gene Expression Regulation, Neoplastic; Gluconates; Glucosamine; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; In Vitro Techniques; Lactic Acid; Leupeptins; Lysosomes; Phosphorylation; Proteasome Endopeptidase Complex; Pyruvic Acid; Signal Transduction; Tongue Neoplasms

To explore the apoptotic effect of ubiquitin-proteasome inhibitor (PI) MG-132 on human tongue squamous cell carcinoma cell line (Tca-8113 cell) through endoplasmic reticulum stress.. Tca-8113 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, the exponential cells were divided into 5 groups.The cell culture medium was added to 1640 (negative control group), thapsigargin 5 μmol/L (positive control group), and 10,20,30 μmol/L (MG-132 group). After 24 h, the following experiments were carried out: morphological change of apoptotic cells was observed by Hoechst 33258 fluorescent staining under fluorescent microscope, apoptotic rate of cells was determined with annexin V-FITC/PI double staining by flow cytometry, the GRP78 mRNA level was determined by RT-PCR, the expression of caspase-12 protein was evaluated by Western blot, the human ubiquitin-protein ligase E3 concentration was detected by ELISA. The data was analyzed using SPSS16.0 software package.. Typical morphological change of apoptosis in Tca-8113 cells was observed 24 hours after treating with 10.0, 20.0, 30.0 μmol/L MG-132 and positive control group; The apoptotic rate of MG-132 groups gradually increased with MG-132 concentration; The GRP78 mRNA level was up-regulated; The expression of caspase-12 increased was demonstrated by Western blot; The expression of the human ubiquitin-protein ligase E3 in MG-132 groups was 28.75 ± 2.28, 18.16 ± 0.65 and 8.85 ± 0.72.. MG-132 can induce apoptosis of Tca-8113 cells through endoplasmic reticulum stress; MG-132 can inhibit the expression of human ubiquitin ligase E3.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Line; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Leupeptins; Thapsigargin; Tongue Neoplasms

The aim of this study was to investigate the apoptotic effect of a proteasome inhibitor MG-132 on Tca-8113, a cell line of human tongue squamous cell carcinoma. Tca-8113 cells were treated with 10, 20, and 30μM of MG-132, or 5μM thapsigargin. Apoptosis rate was determined with annexin V/propidium iodide double staining. Expression of E3ubiquitin-protein ligase was determined by ELISA, and Grp78 and caspase-12 mRNA, and Grp78 and caspase-12 protein was assessed by RT-PCR and Western blot, respectively. Apoptosis was observed 18h after MG-132 treatment. The apoptotic rate in the 10, 20, and 30μM MG-132 group was 13.5, 19.6 and 34.7%, respectively, which was higher than in the thapsigargin (8.5%, P<0.01) or control group (0.5%, P<0.01). The expression of E3 ubiquitin-protein ligase in the 10, 20, and 30μM MG-132 group was 28.75±2.28, 18.16±0.65, 8.85±0.72, respectively, which was lower than in the thapsigargin (38.96±0.33, P<0.05 or 0.01) or control (40.88±4.52, P<0.05 or 0.01) group. The levels of Grp78 and capase-12 mRNA, Grp78 and caspase-12 protein in the MG-132 groups were higher than in the control group (P<0.01). In conclusion, MG-132 induces apoptosis in Tca-8113 cells in a concentration-dependent manner. The MG-132-induced apoptosis may involve downregulation of E3 ubiquitin ligase, and upregulation of Grp78 and caspase-12.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspase 12; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Leupeptins; RNA, Messenger; Thapsigargin; Tongue Neoplasms; Ubiquitin-Protein Ligases