Moon Chang Baek

α-Synuclein Activates Microglia by Inducing the Expressions of Matrix Metalloproteinases and the Subsequent Activation of Protease-Activated Receptor-1

The mutation or overexpression of α-synuclein protein plays a pivotal role in the pathogenesis of Parkinson’s disease. In our preliminary experiments, we found that α-synuclein induced the expression of matrix metalloproteinases (MMPs) (MMP-1, -3, -8, and -9) in rat primary cultured microglia. Thus, the current study was undertaken to determine the roles of MMPs in α-synuclein–induced microglial activation. The inhibition of MMP-3, -8, or -9 significantly reduced NO and reactive oxygen species levels and suppressed the expression of TNF-α and IL-1β. Notably, MMP-8 inhibitor suppressed TNF-α production more efficaciously than MMP-3 or MMP-9 inhibitors. Inhibition of MMP-3 or -9 also suppressed the activities of MAPK, NF-κB, and AP-1. Previously, protease-activated receptor-1 (PAR-1) has been associated with the actions of MMPs, and thus, we further investigated the role of PAR-1 in α-synuclein–induced inflammatory reactions. A PAR-1–specific inhibitor and a PAR-1 antagonist significantly suppressed cytokine levels, and NO and reactive oxygen species production in α-synuclein–treated microglia. Subsequent PAR-1 cleavage assay revealed that MMP-3, -8, and -9, but not α-synuclein, cleaved the synthetic peptide containing conventional PAR-1 cleavage sites. These results suggest that MMPs secreted by α-synuclein–stimulated microglia activate PAR-1 and amplify microglial inflammatory signals in an autocrine or paracrine manner. Furthermore, our findings suggest that modulation of the activities of MMPs and/or PAR-1 may provide a new therapeutic strategy for Parkinson’s disease.

Identification of Developmental Endothelial Locus-1 on Circulating Extracellular Vesicles as a Novel Biomarker for Early Breast Cancer Detection

Purpose: Currently, there are no molecular biomarkers for the early detection of breast cancer. This study focused on identifying surface proteins found on circulating extracellular vesicles (EVs) for detecting early-stage breast cancer. Experimental Design: Circulating EVs, isolated from the plasma of 10 patients with breast cancer (stages I and II) and 5 healthy controls, were analyzed using LC-MS/MS. Developmental endothelial locus-1 protein (Del-1) was selected as a candidate biomarker. Two different ELISAs were used to measure Del-1 in plasma samples from healthy controls (n = 81), patients with breast cancer (n = 269), breast cancer patients after surgical resection (n = 50), patients with benign breast tumors (n = 64), and patients with noncancerous diseases (n = 98) in two cohorts. Results: Plasma Del-1 levels were significantly higher (P < 0.0001) in patients with breast cancer than in all controls and returned to almost normal after tumor removal. The diagnostic accuracy of Del-1 was AUC, 0.961 [95% confidence interval (CI), 0.924–0.983], sensitivity of 94.70%, and specificity of 86.36% in test cohort and 0.968 (0.933–0.988), 92.31%, and 86.62% in validation cohort for early-stage breast cancer by one type of ELISA. Furthermore, Del-1 maintained diagnostic accuracy for patients with early-stage breast cancer using the other type of ELISA [0.946 (0.905–0.972), 90.90%, and 77.14% in the test cohort; 0.943 (0.900–0.971), 89.23%, and 80.99% in the validation cohort]. Conclusions: Del-1 on circulating EVs is a promising marker to improve identification of patients with early-stage breast cancer and distinguish breast cancer from benign breast tumors and noncancerous diseases. Clin Cancer Res; 22(7); 1757–66. ©2015 AACR.

Proteomic analysis of breast cancer tissues to identify biomarker candidates by gel-assisted digestion and label-free quantification methods using LC-MS/MS

This study presents a proteomic method that differentiates between matched normal and breast tumor tissues from ductal carcinoma in situ (DCIS) and invasive carcinoma from Korean women, to identify biomarker candidates and to understand pathogenesis of breast cancer in protein level. Proteins from tissues obtained by biopsy were extracted by RIPA buffer, digested by the gel-assisted method, and analyzed by nano-UPLC-MS/MS. From proteomic analysis based on label-free quantitation strategy, a non-redundant list of 298 proteins was identified from the normal and tumor tissues, and 244 proteins were quantified using IDEAL-Q software. Hierarchical clustering analysis showed two patterns classified as two groups, invasive carcinoma and DCIS, suggesting a difference between two carcinoma at the protein expression level as expected. Differentially expressed proteins in tumor tissues compared to the corresponding normal tissues were related to three biological pathways: antigen-processing and presentation, glycolysis/gluconeogenesis, and complement and coagulation cascades. Among them, the up-regulation of calreticulin (CRT) and protein disulfide isomerase A3 (PDIA3) was confirmed by Western blot analysis. In conclusion, this study showed the possibility of identifying biomarker candidates for breast cancer using tissues and might help to understand the pathophysiology of this cancer at the protein level.

Fibronectin on circulating extracellular vesicles as a liquid biopsy to detect breast cancer

Extracellular vesicles (EVs) secreted from cancer cells have potential for generating cancer biomarker signatures. Fibronectin (FN) was selected as a biomarker candidate, due to the presence in surface on EVs secreted from human breast cancer cell lines. A subsequent study used two types of enzyme-linked immunosorbent assays (ELISA) to determine the presence of these proteins in plasma samples from disease-free individuals (n=70), patients with BC (n=240), BC patients after surgical resection (n=40), patients with benign breast tumor (n=55), and patients with non-cancerous diseases (thyroiditis, gastritis, hepatitis B, and rheumatoid arthritis; n=80). FN levels were significantly elevated (p<. 0001) at all stages of BC, and returned to normal after tumor removal. The diagnostic accuracy for FN detection in extracellular vesicles (ELISA method 1) (area under the curve, 0.81; 95% CI, 0.76 to 0.86; sensitivity of 65.1% and specificity of 83.2%) were also better than those for FN detection in the plasma (ELISA method 2) (area under the curve, 0.77; 95% CI, 0.72 to 0.83; sensitivity of 69.2% and specificity of 73.3%) in BC. The diagnostic accuracy of plasma FN was similar in both the early-stage BC and all BC patients, as well as in the two sets. This liquid biopsy to detect FN on circulating EVs could be a promising method to detect early breast cancer.

Increased liver-specific proteins in circulating extracellular vesicles as potential biomarkers for drug- and alcohol-induced liver injury

Drug- and alcohol-induced liver injury are a leading cause of liver failure and transplantation. Emerging evidence suggests that extracellular vesicles (EVs) are a source of biomarkers because they contain unique proteins reflecting the identity and tissue-specific origin of the EV proteins. This study aimed to determine whether potentially hepatotoxic agents, such as acetaminophen (APAP) and binge alcohol, can increase the amounts of circulating EVs and evaluate liver-specific EV proteins as potential biomarkers for liver injury. The circulating EVs, isolated from plasma of APAP-exposed, ethanol-fed mice, or alcoholic hepatitis patients versus normal control counterparts, were characterized by proteomics and biochemical methods. Liver specific EV proteins were analyzed by immunoblots and ELISA. The amounts of total and liver-specific proteins in circulating EVs from APAP-treated mice significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs from APAP-exposed mice revealed that the amounts of liver-specific and/or hepatotoxic proteins were increased compared to those of controls. Additionally, the increased protein amounts in EVs following APAP exposure returned to basal levels when mice were treated with N-acetylcysteine or glutathione. Similar results of increased amounts and liver-specific proteins in circulating EVs were also observed in mice exposed to hepatotoxic doses of thioacetamide or d-galactosamine but not by non-hepatotoxic penicillin or myotoxic bupivacaine. Additionally, binge ethanol exposure significantly elevated liver-specific proteins in circulating EVs from mice and alcoholics with alcoholic hepatitis, compared to control counterparts. These results indicate that circulating EVs in drug- and alcohol-mediated hepatic injury contain liver-specific proteins that could serve as specific biomarkers for hepatotoxicity.

Enhanced delivery of T cells to tumor after chemotherapy using membrane-anchored, apoptosis-targeted peptide.

Chemotherapy-induced apoptosis of tumor cells enhances the antigen presentation and sensitizes tumor cells to T cell-mediated cytotoxicity. Here we harnessed the apoptosis of tumor cells as a homing signal for the delivery of T cells to tumor. Jurkat T cells were anchored with ApoPep-1, an apoptosis-targeted peptide ligand, using the biocompatible anchor for membrane (BAM), an oleyl acid derivative. The ApoPep-1-BAM conjugate was efficiently anchored to cell membrane, while little anchoring was obtained with ApoPep-1 alone. The retention period of the ApoPep-1-BAM conjugate on cell membrane was approximately 80 and 40 min in the absence and presence of serum, respectively. ApoPep-1 was resistant to degradation in serum until 2h. The apoptosis-targeted T cells that were anchored with the ApoPep-1-BAM preferentially bound to apoptotic tumor cells over living cells. When intravenously injected into tumor-bearing mice, the number of apoptosis-targeted T cells and in vivo fluorescence signals by the homing of the cells to doxorubicin-treated tumor were higher than those of untargeted T cells. Accumulation of apoptosis-targeted T cells at other organs such as liver was not detected. These results suggest that the chemotherapy-induced apoptosis and subsequent enhancement of T cell delivery to tumor by the membrane anchoring of the apoptosis-targeted peptide could be a novel strategy for cancer immunotherapy.

Proteomic Analysis of Pelvic Autonomic Nerve in Nerve-sparing Radical Hysterectomy for Cervical Carcinoma

Background/Aim: This study was designed to identify candidate proteins which can be used for visualization of pelvic autonomic nerve during nerve-sparing surgery. Materials and Methods: Both soft tissue and vesical branch of the inferior hypogastric nerve from five women were collected during surgery. These 10 tissue specimens were analysed using liquid chromatography–mass spectrometry (LC-MS) and Human Protein Atlas (HPA) for protein expression. The existence of nerve fibres was confirmed using haematoxylin and eosin (H&E) and anti-S-100 staining. Results: A total of 413 proteins were detected. There were three proteins (isoform 1 of fibronectin, protein S100-A8 and A9) which implied a relation with pelvic autonomic nerve. In nerve tissue from one case, the existence of nerve fibre was not confirmed. Conclusion: Further large studies are expected to present more nerve-specific candidate proteins which can be used for the easy and safe identification of autonomic nerves.