ABSTRACT
The metalloproteinase ADAM10 critically contributes to development, inflammation, and cancer and can be controlled by endogenous or synthetic inhibitors. Here, we demonstrate for the first time that loss of proteolytic activity of ADAM10 by either inhibition or loss of function mutations induces removal of the protease from the cell surface and the whole cell. This process is temperature dependent, restricted to mature ADAM10, and associated with an increased internalization, lysosomal degradation, and release of mature ADAM10 in extracellular vesicles. Recovery from this depletion requires de novo synthesis. Functionally, this is reflected by loss and recovery of ADAM10 substrate shedding. Finally, ADAM10 inhibition in mice reduces systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. These findings are crucial for development of therapeutic ADAM10 inhibition strategies and may showcase a novel, physiologically relevant mechanism of protease removal due to activity loss.
Subject(s)
ADAM10 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Extracellular Vesicles/metabolism , Membrane Proteins/metabolism , ADAM10 Protein/analysis , ADAM10 Protein/genetics , Amyloid Precursor Protein Secretases/analysis , Amyloid Precursor Protein Secretases/genetics , Animals , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Extracellular Vesicles/genetics , Humans , Loss of Function Mutation , Male , Membrane Proteins/analysis , Membrane Proteins/genetics , Mice, Inbred C57BL , ProteolysisABSTRACT
There is a strong demand for bioanalytical techniques to rapidly detect protease activities with high sensitivity and high specificity. This study reports an activity-based electrochemical method toward this goal. Nanoelectrode arrays (NEAs) fabricated with embedded vertically aligned carbon nanofibers (VACNFs) are functionalized with specific peptide substrates containing a ferrocene (Fc) tag. The kinetic proteolysis curves are measured with continuously repeated ac voltammetry, from which the catalytic activity is derived as the inverse of the exponential decay time constant based on a heterogeneous Michaelis-Menten model. Comparison of three peptide substrates with different lengths reveals that the hexapeptide H2N-(CH2)4-CO-Pro-Leu-Arg-Phe-Gly-Ala-NH-CH2-Fc is the optimal probe for cathepsin B. The activity strongly depends on temperature and is the highest around the body temperature. With the optimized peptide substrate and measuring conditions, the limit of detection of cathepsin B activity and concentration can reach 2.49 × 10-4 s-1 and 0.32 nM, respectively. The peptide substrates show high specificity to the cognate proteases, with negligible cross-reactions among three cancer-related proteases cathepsin B, ADAM10, and ADAM17. This electrochemical method can be developed into multiplex chips for rapid profiling of protease activities in cancer diagnosis and treatment monitoring.
Subject(s)
ADAM10 Protein/analysis , ADAM17 Protein/analysis , Amyloid Precursor Protein Secretases/analysis , Carbon/chemistry , Cathepsin B/analysis , Electrochemical Techniques/methods , Electrodes , Membrane Proteins/analysis , Nanofibers/chemistry , ADAM10 Protein/metabolism , ADAM17 Protein/metabolism , Amyloid Precursor Protein Secretases/metabolism , Cathepsin B/metabolism , Humans , Membrane Proteins/metabolism , Nanotechnology , ProteolysisABSTRACT
ADAMs (a disintegrin and metalloproteinase) are important mediators of cell signalling events, which play a role in the pathogenesis and progression of cancers. Immunohistochemical method was used to examine the immunoexpression of ADAM10 and microvessel density in 80 cases of oral squamous cell carcinoma (OSCC): without metastases - OSCC M(-) (n = 38), and with metastases - OSCC M(+) (n = 42), in 24 cases of oral leukoplakia (OLK), (15 cases with low-grade dysplasia - OLK-LG, and 9 cases with high-grade dysplasia - OLK-HG), and 19 controls. The immunoexpression of ADAM10 and the mean number of vessels were significantly increased in both groups of OSCC in comparison to both groups of OLK and controls. Moreover, the immunoexpression of ADAM10 and microvessel density were significantly increased in the OSCC M(+) group in comparison to the OSCC M(-) group. No statistically significant differences were found between immunoexpression of ADAM10 and microvessels density in the OLK-LG, OLK-HG, and control cases. In conclusion, the present study revealed overexpression of ADAM10 in OSCCs, especially in OSCC with metastasis. These findings suggest that ADAM10 could potentially contribute to metastases of oral cancer. Although, our findings suggest that ADAM10 may be involved in angiogenesis of OSCC, further studies are required to determine the role of ADAM10 in this process.
Subject(s)
ADAM10 Protein/analysis , Amyloid Precursor Protein Secretases/analysis , Biomarkers, Tumor/analysis , Carcinoma, Squamous Cell/enzymology , Head and Neck Neoplasms/enzymology , Membrane Proteins/analysis , Mouth Neoplasms/enzymology , Adult , Aged , Aged, 80 and over , Biopsy , Capillaries/enzymology , Capillaries/pathology , Carcinoma, Squamous Cell/secondary , Female , Head and Neck Neoplasms/pathology , Humans , Immunohistochemistry , Leukoplakia, Oral/enzymology , Leukoplakia, Oral/pathology , Male , Middle Aged , Mouth Neoplasms/pathology , Neovascularization, Pathologic , Retrospective Studies , Squamous Cell Carcinoma of Head and Neck , Up-RegulationABSTRACT
Due to early onset of local invasion and distant metastasis, pancreatic cancer is the most lethal human malignant tumor, with a 5 year survival rate of less than 5%. As a effective chemotherapy drug for pancreatic cancer patients, gemcitabine is reported to inhibit cell proliferation as a nucleotide analog. In this study, we investigated the role of gemcitabine in immune regulation of pancreatic cancer. Our data showed that the level of soluble ULBP2 (sULBP2), a ligand of NKG2D receptor, decreased in the supernatants of pancreatic cancer cell lines when gemcitabine was added, and sULBP2 level correlated with NK92 cells cytotoxicity to pancreatic cancer cell lines. Importantly, our data showed that gemcitabine promoted PANC-1 cells and MIA PaCa-2 immune evasion by reducing ADAM10 expression, a metalloproteinase involved in sULBP2 shedding from cell membrane. Knockdown of ADAM10 clearly downregulated sULBP2 levels in the culture supernatants and cells became more susceptible to NK92 cytotoxicity. Serum samples and tumor samples were obtained from 45 patients with pancreatic ductal adenocarcinoma (PDAC). Statistical analysis showed a significant correlation between the serum level of sULBP2 with ADAM10 expression in PDAC tissues. In conclusion, our data demostrated that gemcitabine inhibits ULBP2 ectodomain shedding through the suppression of ADAM10 and enhance NK cells cytotoxicity by NKG2D-ULBP2 interaction. The results extends our understanding of gemcitabine in the treatment of pancreatic cancer from cell proliferation inhibition to immune regulation.