leupeptins and Mouth-Neoplasms

Epidermal growth factor receptor (EGFR) is highly expressed in head and neck squamous cell carcinoma (HNSCC) and correlates with poor prognosis. EGFR has been demonstrated to be associated with cancer stem cell traits in HNSCC. However, the underlying molecular mechanism is far from elucidated. Here, SOX2, one of the most important stem cell markers, was identified as a binding partner and substrate of EGFR. EGFR signaling inhibition decreases SOX2 expression by promoting its autophagic degradation. Mechanistically, EGFR activation induces SOX2 phosphorylation at the Y277 site and reduces its ubiquitination, which inhibits its association with p62 and subsequent autophagic degradation. Gefitinib, an EGFR tyrosine kinase inhibitor, shows in vitro and in vivo protective effects against oral cancer cells that can be reversed through autophagy inhibition. Our study suggests that EGFR plays an important role in the development of cancer stem cells by stabilizing SOX2. Targeting EGFR in combination with conventional chemotherapy might be a promising strategy for the treatment of HNSCC through elimination of cancer stem cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Autophagy; Cell Line, Tumor; Cell Proliferation; ErbB Receptors; Gefitinib; Humans; Leupeptins; Macrolides; Male; Mice; Mouth Neoplasms; Mutagenesis; Neoplastic Stem Cells; Protein Kinase Inhibitors; Protein Stability; Proteolysis; SOXB1 Transcription Factors; Squamous Cell Carcinoma of Head and Neck; Xenograft Model Antitumor Assays

Proteasome inhibitor Carbobenzoxy-Leu-Leu-leucinal (MG132) induces the unfolded protein response (UPR) in oral squamous cell carcinoma (OSCC). X-box binding protein 1 (XBP1) is a key UPR component that regulates endoplasmic reticulum stress (ER) homeostasis. This study was aimed to investigate the activation of IRE1α-TRAF2-ASK1-JNK pathway by silencing the XBP1 expression in an OSCC cell line.. The XBP1 specific short hairpin RNA (shRNA) plasmid vector was constructed and then transfected into the Tca-8113 cells. The effect of XBP-1 gene silencing on IRE1α-TRAF2-ASK1-JNK pathway under MG132 induced endoplasmic reticulum stress in Tca-8113 were investigated by real-time RT-PCR or western blot. Cell apoptosis was detected by flow cytometry.. XBP1 expression was reduced in transfected groups and MG132 groups. shRNA-XBP1 induces IRE1α-TRAF2-ASK1 signaling activation to activate pro-apoptotic ASK1-JNK signaling. Moreover, combined shRNA-XBP1 with MG132 further enhanced downregulated XBP1 expression and upregulated activation of ASK1-JNK signaling.. Silencing XBP1 expression under MG132 induced ER stress block the XBP1 survival pathway and synergism with MG132 to promote Tca8113 cell apoptosis. These findings provide a therapeutic option in oral squamous cell carcinoma by inhibition of proteasome and XBP1 splicing.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; DNA-Binding Proteins; Endoplasmic Reticulum Stress; Endoribonucleases; Gene Silencing; Humans; Leupeptins; MAP Kinase Signaling System; Mouth Neoplasms; Protein Serine-Threonine Kinases; Regulatory Factor X Transcription Factors; RNA, Small Interfering; Signal Transduction; TNF Receptor-Associated Factor 2; Transcription Factors; X-Box Binding Protein 1

E-cadherin/β-catenin complex plays an important role in maintaining the homeostasis of tissues and regulating cell proliferation, survival and apoptosis. To address the relationships between the change of E-cadherin/β-catenin complex and cell apoptosis, human oral squamous carcinoma SCC-25 cells were used to investigate whether the dissociation of the E-cadherin/β-catenin complex was the main reason of MG132- or bortezomib-induced apoptosis. We found that MG132 or bortezomib alone induced remarkable loss of cell integrity and contact, inhibited cell growth, survival, migration and caused cell cycle arrest, intracellular ROS production. Further experiments showed that colony formations were significantly decreased by MG132 and bortezomib alone or plus cis-diaminedichloroplatinum (CDDP). Immunofluorescence staining showed that SCC-25 cells exhibited remarkable accumulations of β-catenin in cytoplasm and few E-cadherin in cell membranes after MG132 or bortezomib treatment. Western blot results showed that MG132 or bortezomib induced high accumulation of ubiquitinated proteins and activation of apoptosis related protein caspase-3. Meanwhile, the combinational use of MG132 or bortezomib with CDDP led to synergistic effects on SCC-25 cells. However, knockdown of β-catenin could decrease MG132 or bortezomib induced cell death. Taken together, our data suggest that the regulation of E-cadherin/β-catenin complex could be a promising therapeutic target to overcome the multidrug resistance of oral cancer.

Topics: beta Catenin; Bortezomib; Cadherins; Cell Adhesion; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Movement; Cisplatin; DNA Breaks; Drug Resistance, Neoplasm; Humans; Leupeptins; Mouth Neoplasms

Oral squamous cell carcinoma (OSCC) cells are relatively resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis during culture. We investigated the role of a proteaosome inhibitor in the survival and apoptosis of these cells. We found that the proteasome inhibitor MG132 markedly accelerated TRAIL-mediated apoptosis in OSCC cell lines HSC-2 and HSC-3. Addition of TRAIL to MG132-treated cells resulted in Bid cleavage. Furthermore, the inhibitors of caspase-3, caspase-8 and caspase-9 reduced the accelerative effect of MG132 on TRAIL-mediated apoptosis. These results suggest that the pro-apoptotic effect of a proteasome inhibitor on TRAIL-mediated apoptosis may contribute to both extrinsic and intrinsic pathways. MG132 enhanced the expression of the TRAIL receptors DR4 and DR5, and neutralization of DR5 receptors showed a marked reduction of TRAIL-mediated apoptosis, whereas that of DR4 was a partial reduction. MG132 also markedly reduced cellular FLICE-inhibitory protein (c-FLIP), cellular inhibitor of apoptosis protein-1 (cIAP-1), X-linked IAP (XIAP) and survivin. Therefore, MG132 provides partial regulation of TRAIL-mediated apoptosis in OSCC cells via modulation of DR5, c-FLIP, cIAP-1, XIAP and survivin. The proteasome inhibitor MG132 may therefore represent a novel strategy for overcoming resistance to TRAIL-mediated apoptosis in OSCC cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Drug Synergism; Humans; Leupeptins; Mouth Neoplasms; Proteasome Inhibitors; TNF-Related Apoptosis-Inducing Ligand

Ubiquitin-mediated proteolysis controls intracellular levels of various cell cycle regulatory proteins, and its inhibition has been shown to induce apoptosis in proliferating cells. In the present study, we examined induction of apoptosis in oral squamous cell carcinoma (OSCC) cells by treatment with specific proteasome inhibitors, carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal and lactacystin. In all three OSCC cell lines examined, apoptotic changes such as apoptotic body formation and DNA fragmentation were observed at various degrees after 24 h of the carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal or lactacystin treatment. HSC2 cells showed the most prominent apoptotic changes among the cell lines examined and demonstrated the highest level of accumulation of p27Kip1 protein after the treatment with proteasome inhibitor. Reduced expressions of cyclin D1 and phospho pRb were also observed after the treatment with proteasome inhibitor. Moreover, 12 h of treatment with the proteasome inhibitor inhibited cdk2/cyclin E kinase activity and increased the ratio of the cell cycle population at the G1 phase. The proteasome inhibitor led to inhibition of cell cycle progression. In addition, activation of CPP32 and reduced expression of Bcl-2 were observed. Because apoptosis induced by the proteasome inhibitor was inhibited by treatment with antisense p27Kip1 oligonucleotide, accumulation of the p27Kip1 protein might play an important role in the apoptosis induced by proteasome inhibitor. The present results suggest that inhibition of proteasome function may be used as a possible target of novel therapy for OSCC.

Topics: Acetylcysteine; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line; Cyclin-Dependent Kinase Inhibitor p27; Cysteine Endopeptidases; Humans; Leupeptins; Microtubule-Associated Proteins; Mouth Mucosa; Mouth Neoplasms; Multienzyme Complexes; Oligonucleotides, Antisense; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Precursors; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Tumor Suppressor Proteins

We investigated whether proteasomes were involved in the invasiveness of oral squamous cell carcinoma (SCC) cells. The migration of SCC cells through a gelatin-coated membrane was enhanced with tumor necrosis factor alpha (TNF alpha), which was strongly inhibited by a peptide aldehyde, N-acetyl-Leu-Leu-norleucinal (ALLN), but not by its structurally related compound, N-acetyl-Leu-Leu-methioninal (ALLM). Since ALLN is a more potent inhibitor against proteasomal proteolysis than ALLM, cell migration inhibited by ALLN may thus likely depend on proteasomes. The TNF alpha-induced migration through gelatin appeared to be associated with the gelatinolytic activity from the cells, since TNF alpha strongly enhanced the production of matrix metalloproteinase (MMP)-9/gelatinase B in the SCC cells, as detected by gelatin zymography. The production of MMP-9 was also inhibited by pretreatment with ALLN, but not ALLM, in a dose-dependent manner. Moreover, ALLN could block the activation and nuclear translocation of a transcription-activating factor, NF-kappaB, which is known to regulate MMP-9 expression in TNF alpha-stimulated SCC cells. The TNF alpha-induced degradation of IkappaB alpha was also suppressed by ALLN treatment, thus implying that the molecule linking proteasome to MMP-9 production should be IkappaB alpha. We finally reconfirmed the involvement of proteasomes in the invasive behavior of oral SCC using lactacystin, a specific proteasome inhibitor, which could prevent TNF alpha from enhancing MMP-9 production, NF-kappaB activation, induction of MMP-9 mRNA and cell migration.

Topics: Carcinoma, Squamous Cell; Collagenases; Cysteine Proteinase Inhibitors; Gelatinases; Humans; Leupeptins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins; NF-kappa B; Oligopeptides; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Reduced expression of the cyclin-dependent kinase inhibitor p27Kip1 has been reported to correlate with poor survival in cohorts of breast and colorectal carcinoma patients. Posttranslational ubiquitin-mediated proteasomal proteolysis is related to p27Kip1 protein levels. However, to the authors' knowledge, no previous study has examined the expression of p27Kip1 in oral squamous cell carcinoma (OSCC).. To examine the expression of p27Kip1 and its clinicopathologic roles in OSCC, the authors studied the expression of p27Kip1 protein by immunohistochemistry in deparaffinized tissue sections of 20 normal oral mucosa specimens, 22 epithelial dysplasia specimens, and 70 OSCCs, and analyzed its correlation with clinicopathologic parameters. They also studied the expression of p27Kip1 mRNA and protein in six OSCC cell lines by Northern blot and Western blot analysis. To examine the mechanism of reduced expression of p27Kip1, OSCC cell lines were treated with the proteasome inhibitor LLnV.. All the normal oral mucosa specimens and 73% (16 of 22) of the oral epithelial dysplasia specimens expressed p27Kip1 at high levels, whereas 87% of the OSCCs (61 of 70) showed reduced expression of p27Kip1. Furthermore, the levels of expression of this protein were significantly lower in carcinomas with metastasis than those without metastasis. Although OSCC cell lines expressed p27Kip1 mRNA at various levels, most of them expressed p27Kip1 protein at lower or undetectable levels. LLnV induced the expression of p27Kip1 protein in HSC2 cells, in which p27Kip1 protein was originally undetectable.. These findings suggest that 1) reduced expression of p27Kip1 may correlate with the development and progression of OSCC and can be an indicator of malignant behavior of this neoplasm, and 2) increased proteasome-mediated degradation may play an important role in the reduction of p27Kip1 protein expression.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Cysteine Proteinase Inhibitors; Disease Progression; Female; Humans; Leupeptins; Male; Microtubule-Associated Proteins; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Neoplasm Proteins; Oligopeptides; Prognosis; Tumor Cells, Cultured; Tumor Suppressor Proteins

Protein phosphorylation was studied in crude and in protein kinase C (Pk-C)-enriched preparations from squamous cell carcinomas and normal mucosa of the human upper aero-digestive tract. In crude soluble preparations from neoplastic mucosa we found a 5-fold higher basal endogenous phosphorylation when compared to normal mucosa. In particulate fractions the increase was 3-fold. SDS-PAGE and autoradiography of phosphorylated proteins in crude soluble tumor extracts showed bands corresponding to proteins with apparent molecular weights of 18, 37, 40-42, 52, 60, 62 and 90 kDa. In normal mucosa the phosphorylation of these proteins was very low or absent, except for the proteins with molecular weights of 40-42 and 52-55 kDa. Addition of Ca2+ or Ca2+/phospholipids to the reaction mixture caused phosphorylation of additional proteins with apparent molecular weight of 45-50 kDa in soluble preparations of tumors. Cyclic AMP or cGMP had no significant effect on the phosphorylation of endogenous proteins. In the partially purified, Pk-C-enriched fractions the phosphorylation in the presence of Ca2+/phospholipids was distinctly higher in tumors when compared to the phosphorylation observed in normal mucosa, and some phosphorylation substrates were detected only in tumor tissue. In order to find out whether the elevated basal phosphorylation was due to an endogenous activation of protein kinases, different inhibitors of serine/threonine protein kinases were tested. These inhibitors included: heat-stable cyclic AMP-dependent protein kinase (Pk-A) inhibitor, Pk-A inhibitor peptide (Wiptide), heparin and the Pk-C inhibitors peptide 19-36 and H-7. None of these inhibitors had any significant effect on the basal phosphorylation. In conclusion, our results show the existence of endogenous phosphorylation substrates in human squamous cell carcinomas from the upper aerodigestive tract, and indicates that there is a significantly higher basal and Pk-C specific phosphorylation of endogenous substrates in tumors compared to normal mucosa. This may be of importance for the transformation and altered growth regulation in epithelial tumors.

Topics: Aged; Autoradiography; Calcium; Carcinoma, Squamous Cell; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Humans; Leupeptins; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Neoplasm Proteins; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Proteins; Tissue Extracts