leupeptins and Esophageal-Neoplasms

It has been implied that deregulation of cyclin D1 turnover under stresses can facilitate genomic instability and trigger tumorigenesis. Much focus has been placed on identifying the E3 ligases responsible for mediating cyclin D1 degradation. However, the findings were quite controversial and cell type-dependent. Little is known about how cyclin D1 is regulated in precancerous cells upon DNA damage and which E3 ligases mediate the effects. Here we found cyclin D1 reduction is an early response to DNA damage in immortalized esophageal epithelial cells, with expression dropping to a low level within 1 h after γ-irradiation. Comparison of temporal expression of cyclin D1 upon DNA damage between immortalized NE083-hTERT and NE083-E6E7, the latter being p53/p21-defective, showed that DNA damage-induced rapid cyclin D1 reduction was p53-independent and occurred before p21 accumulation. Overexpression of cyclin D1 in NE083-E6E7 cells could attenuate G0/G1 cell cycle arrest at 1 h after irradiation. Furthermore, rapid reduction of cyclin D1 upon DNA damage was attributed to proteasomal degradation, as evidenced by data showing that proteasomal inhibition by MG132 blocked cyclin D1 reduction while cycloheximide facilitated it. Inhibition of ATM activation and knockdown of E3 ligase adaptor FBX4 reversed cyclin D1 turnover in immortalized NE083-hTERT cells. Further study showed that knockdown of FBX4 facilitated DNA breaks, as indicated by an increase in γ-H2AX foci in esophageal cancer cells. Taken together, the results substantiated a pivotal role of ATM and FBX4 in cyclin D1 proteolysis upon DNA damage in precancerous esophageal epithelial cells, implying that deregulation of the process may contribute to carcinogenesis of esophageal squamous cell carcinoma.

Topics: Cell Cycle; Cyclin D1; Cycloheximide; DNA Damage; Down-Regulation; Epithelial Cells; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Esophagus; F-Box Proteins; Gamma Rays; Humans; Leupeptins; Proteasome Endopeptidase Complex; Proteolysis; Tumor Suppressor Protein p53

We recently reported that STAT1 plays a tumor suppressor role, and ERK was inversely correlation with STAT1 expression in esophageal squamous cell carcinoma (ESCC). Here, we investigated the mechanism(s) that are responsible for the ERK regulates STAT1 in ESCC.. We performed the immunoprecipitation (IP) to detect the ubiquitin of STAT1 upon MEK transfection or U0126 treatment and co-IP to confirm the binding of STAT1 and ERK in ESCC cell lines.. We found evidence that the ubiquitin-proteasome pathway can efficiently degrade STAT1 in ESCC cells, as MG132 treatment rapidly and dramatically increased STAT1 expression in these cells. This process is not dependent on the phosphorylation of the two important STAT1 residues, Y701 and S727, as site-directed mutagenesis of these two sites did not affect STAT1 degradation. We also found that ERK promotes proteasome degradation of STAT1, supported by the observations that pharmacologic inhibition of ERK resulted in a substantial increase of STAT1 whereas expression of constitutively active ERK further reduced the STAT1 protein level. In addition to suppressing STAT1 expression, ERK limited STAT1 signaling by decreasing the production of IFNγ.. To conclude, ERK is an effective negative regulator of STAT1 signaling in ESCC, by promoting its proteasome degradation and decreasing IFNγ production. Our data further supports that targeting ERK and/or STAT1 may be useful for treating ESCC.

Topics: Apoptosis; Cell Line, Tumor; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Extracellular Signal-Regulated MAP Kinases; Feedback, Physiological; Humans; Interferon-gamma; Leupeptins; Phosphorylation; Proteasome Endopeptidase Complex; Proteolysis; RNA, Small Interfering; Signal Transduction; STAT1 Transcription Factor; Ubiquitination

To explore whether MG-132 could enhance the anti-tumor activity of obatoclax against esophageal cancer cell line CaES-17.. MTT assay was used to determine the cytotoxicity of obatoclax and MG-132 in CaES-17 cells. The IC(50) of obatoclax and MG-132 were used to determine the molar ratio (1:2.4) of the two drugs for combined treatment of the cells. The concentrations of obatoclax and MG-132 ranged from 1/8 IC(50) to 4 IC(50) after serial dilution, and their combination index (CI) was calculated using CompuSyn software. The expression of ubiquitin and the cleavage of PARP, caspase-9, phospho-histone H3 and phospho-aurora A/B/C in the exposed cells were examined with Western blotting; the cell apoptosis was measured by flow cytometry with Annexin V staining, and the percentage of cells in each cell cycle phase was also determined by flow cytometry.. The CI of obatoclax and MG-132 was 0.296 for a 50% inhibition of Caes-17 cells and was 0.104 for a 95% inhibition. The cells treated with obatoclax or MG-132 alone showed increased expression of ubiquitin and cleavage of PARP and caspase-9. Compared with the cells treated with obatoclax or MG-132 alone, the cells with a combined treatment exhibited significantly increased expression of ubiquitin, cleavage of PARP and caspase-9, and expression of phospho-Histone H3 (P<0.05). The combined treatment of the cells also resulted in significantly increased expression of phospho-Aurora A/B/C compared with obatoclax treatment alone. The cells with the combined treatment showed significantly higher percentages of apoptotic cells and cells in sub-G(1) and G(2)/M phases compared with the cells treated with either of the drugs (P<0.05).. Obatoclax combined with MG-132 shows a significant synergistic anti-tumor effect against esophageal cancer CaES-17 cells by inducing apoptosis and cell cycle arrest.

Topics: Apoptosis; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Esophageal Neoplasms; Histones; Humans; Indoles; Leupeptins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Pyrroles

Comprehensive treatment based on chemotherapy is regarded as the first-line treatment for patients with unresectable or metastatic esophageal squamous cell carcinoma (ESCC). However, chemoresistance is common among patients with ESCC. Therefore, there is a need to explore new therapeutic strategies or adjuvant drugs. One promising possibility is to use dietary agents that can increase tumor cell sensitivity to drugs. In this study, we initially investigated the antitumor activity of proteasome inhibitor MG132 in vitro and in vivo. Effects of MG132 on the enhancment of the anticancer functions of cisplatin were then investigated in human esophageal cancer EC9706 cells in relation to apoptosis and cell signaling events. Exposure of cells to MG132 resulted in a marked decrease in cell viability in a dose- and time-dependent manner. Administration of MG132 markedly inhibited tumor growth in the EC9706 xenograft model. MG132 significantly enhanced cisplatin-induced apoptosis in association with the activation of caspase-3 and -8. These events were accompanied by the downregulation of NF-κB, which plays a key role in cell apoptosis. Taken together, these findings demonstrate a novel mechanism by which proteasome inhibitor MG132 potentiates cisplatin-induced apoptosis in human ESCC and inhibitory activity of tumor growth of the EC9706 xenograft model.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Cysteine Proteinase Inhibitors; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Humans; Leupeptins; Mice; Mice, Nude

The correlation of S-phase kinase-associated protein 2 (Skp2) with metastasis and prognosis in esophageal squamous cell carcinoma (ESCC) is controversial. The purpose of this study was to explore whether there was a correlation between the expression of Skp2 evaluated by immunohistochemistry and the clinical outcome of patients with operable ESCC, and to further determine the possible mechanism of the impact of Skp2 on survival.. Tissue microarrays that included 157 surgically resected ESCC specimens was successfully generated for immunohistochemical evaluation. The clinical/prognostic significance of Skp2 expression was analyzed. Kaplan-Meier analysis was used to compare the postoperative survival between groups. The prognostic impact of clinicopathologic variables and Skp2 expression was evaluated using a Cox proportional hazards model. A cell proliferation assay and a colony formation assay were performed in ESCC cell lines to determine the function of Skp2 on the progression of ESCC in vitro.. Skp2 expression correlated closely with the T category (p = 0.035) and the pathological tumor-node-metastasis (TNM) stage (p = 0.027). High expression of Skp2 was associated with poor overall survival in resectable ESCC (p = 0.01). The multivariate Cox regression analysis demonstrated that pathological T category, pathological N category, cell differentiation, and negative Skp2 expression were independent factors for better overall survival. In vitro assays of ESCC cell lines demonstrated that Skp2 promoted the proliferative and colony-forming capacity of ESCCs.. Negative Skp2 expression in primary resected ESCC is an independent factor for better survival. Skp2 may play a pro-proliferative role in ESCC cells.

Topics: Aged; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cohort Studies; Esophageal Neoplasms; Female; Gene Knockdown Techniques; Humans; Leupeptins; Male; Middle Aged; Multivariate Analysis; Proteasome Endopeptidase Complex; Proteasome Inhibitors; S-Phase Kinase-Associated Proteins; Survival Analysis; Tumor Stem Cell Assay

p63 is a member of the p53 protein family that regulates differentiation and morphogenesis in epithelial tissues and is required for the formation of squamous epithelia. Barrett's mucosa is a glandular metaplasia of the squamous epithelium that develops in the lower esophagus in the context of chronic, gastroesophageal reflux and is considered as a precursor for adenocarcinoma. Normal or squamous cancer esophageal cells were exposed to deoxycholic acid (DCA, 50, 100, or 200 microM) and chenodeoxycholic and taurochenodeoxycholic acid at pH 5. p63 and cyclooxygenase-2 (COX-2) expressions were studied by Western blot and RT-PCR. DCA exposure at pH 5 led to a spectacular decrease in the levels of all isoforms of the p63 proteins. This decrease was observed within minutes of exposure, with a synergistic effect between DCA and acid. Within the same time frame, levels of p63 mRNA were relatively unaffected, whereas levels of COX-2, a marker of stress responses often induced in Barrett's mucosa, were increased. Similar results were obtained with chenodeoxycholic acid but not its taurine conjugate at pH 5. Proteasome inhibition by lactacystin or MG-132 partially blocked the decrease in p63, suggesting a posttranslational degradation mechanism. These results show that combined exposure to bile salt and acid downregulates a critical regulator of squamous differentiation, providing a mechanism to explain the replacement of squamous epithelium by a glandular metaplasia upon exposure of the lower esophagus to gastric reflux.

Topics: Acetylcysteine; Apoptosis; Barrett Esophagus; Carcinoma, Squamous Cell; Cell Line, Tumor; Cells, Cultured; Chenodeoxycholic Acid; Cyclooxygenase 2; Deoxycholic Acid; DNA-Binding Proteins; Down-Regulation; Doxorubicin; Esophageal Neoplasms; Esophagus; Fluorescent Antibody Technique; Humans; Hydrogen-Ion Concentration; Leupeptins; Proteasome Inhibitors; Taurochenodeoxycholic Acid; Trans-Activators; Transcription Factors; Tumor Suppressor Proteins

To investigate the inhibitory effect of ubiquitin-proteasome pathway (UPP) on proliferation of esophageal carcinoma cells.. Esophageal carcinoma cell strain EC9706 was treated with MG-132 to inhibit its UPP specificity. Cell growth suppression was evaluated with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA synthesis was evaluated by (3)H-thymidine ((3)H-TdR) incorporation. Morphologic changes of cells were observed under microscope. Activity of telomerase was examined by telomeric repeat amplification protocol (TRAP) of PCR-ELISA. Cell cycle and apoptosis were detected by flow cytometry (FCM). DNA fragment analysis was used to confirm the presence of apoptosis. Expression of p27(kip1) was detected by immunocytochemical technique.. After exposed to MG-132, the growth and value of (3)H-TdR incorporation of EC9706 cells were obviously inhibited. Cells became round, small and exfoliative under microscope. TRAP PCR-ELISA showed that light absorption of cells gradually decreased after exposed to 5 micromol/L of MG-132 for 24, 48, 72 and 96 h (P<0.01). The percentage of cells at G(0)/G(1) phase was increased and that at S and G(2)/M was decreased (P<0.01). The rate of apoptotic cells treated with 5 micromol/L of MG-132 for 48 and 96 h was 31.7% and 66.4%, respectively. Agarose electrophoresis showed marked ladders. In addition, the positive signals of p27(kip1) were located in cytoplasm and nuclei in MG-132 group in contrast to cytoplasm staining in control group.. MG-132 can obviously inhibit proliferation of EC9706 cells and induce apoptosis. The mechanisms include upregulation of p27(kip1) expression, G(1) arrest and depression of telomerase activity. The results indicate that inhibiting UPP is a novel strategy for esophageal carcinoma therapy.

Topics: Antineoplastic Agents; Cell Division; Cell Line, Tumor; Esophageal Neoplasms; Humans; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Ubiquitin

EphA2 is a member of the Eph family of receptor tyrosine kinases, which interact with cell-bound ligands known as ephrins. EphA2 expression was investigated by immunohistochemistry with an anti-EphA2 monoclonal antibody in 80 patients with esophageal squamous cell carcinoma (ESCC) who had undergone surgery. EphA2 overexpression was positive in 40 of the 80 patients (50%). A significant correlation was observed between EphA2 expression and regional lymph node metastasis (p=0.023), number of lymph node metastases (p=0.011) and poor degree of tumor differentiation (p=0.004). The survival rates of EphA2-positive patients were poorer than those of EphA2-negative patients (p=0.014). The 5-year survival rate of patients without EphA2 overexpression was 68%, whereas that of patients with EphA2 overexpression was 29%. EphA2 expression was also investigated in 7 ESCC cell lines (TE-1, -2, -8, -13, -15, TT and TTn) and 1 immortalized human esophageal keratinocyte cell line (CHEK-1). Western blotting revealed different levels of EphA2 expression in the 8 cell lines. EphA2 was expressed at a high level in the ESCC cell lines compared to CHEK-1. EphA2 phosphorylation was demonstrated in all cell lines. Northern blot analysis showed that EphA2 mRNA expression in TE-1 was greater than that in the other ESCC cell lines. The observation of small gaps on Western blot analysis of the ESCC cell lines suggests that there may be a mechanism for EphA2 regulation at the point of translation. In conclusion, EphA2 overexpression appears to be related to poor degree of tumor differentiation and lymph node metastasis in ESCC. Consequently, patients with EphA2 overexpression have a poorer prognosis than those without. EphA2 is a potential target to prevent ESCC cells spreading into the lymphatic drainage.

Topics: Adult; Aged; Blotting, Northern; Blotting, Western; Carcinoma, Squamous Cell; Cysteine Proteinase Inhibitors; DNA, Neoplasm; Ephrin-A1; Esophageal Neoplasms; Female; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Leupeptins; Lymphatic Metastasis; Male; Middle Aged; Mutation; Phosphorylation; Prognosis; Protein-Tyrosine Kinases; Receptor, EphA2; RNA, Neoplasm; Survival Rate; Tumor Cells, Cultured