Proteomic analysis of human umbilical cord serum exosomes using mass spectrometry and preliminary study of their biological activities in liver cancer cell lines.

Exosomes are membranous extracellular vesicles 50-100 nm in size, which are involved in cellular communication via the delivery of proteins, lipids and RNA. Emerging evidence shows that exosomes play a critical role in cancer. It has recently been revealed that maternal and umbilical cord serum (UCS)-derived exosomes may enhance endothelial cell proliferation and migration. However, the role of exosomes isolated from the human umbilical cord in cancer development has not been investigated. To explore the potential differences in the composition and function of proteins from umbilical serum exosomes (UEs) and maternal serum exosomes, a proteomic analysis of exosomes was conducted using mass spectrometry and bioinformatics. Moreover, Cell Counting Kit-8 assays and flow cytometry were used to study the biological effects of UEs on liver cancer cell lines. The present study demonstrated that UCS was enriched with proteins involved in extracellular matrix-receptor interactions, which may be closely related to cell metastasis and proliferation. The findings further indicated that exosomes derived from human umbilical serum could inhibit the viability and induce apoptosis of liver cancer cells. This suggests that UCS-derived exosomes may represent potential leads for the development of biotherapy for liver cancer.

Potent antitumor activity of a glutamyltransferase-derived peptide via an activation of oncosis pathway.

Hepatocellular carcinoma (HCC) still presents poor prognosis with high mortality rate, despite of the improvement in the management. The challenge for precision treatment was due to the fact that little targeted therapeutics are available for HCC. Recent studies show that metabolic and circulating peptides serve as endogenous switches for correcting aberrant cellular plasticity. Here we explored the antitumor activity of low molecular components in human umbilical serum and identified a high abundance peptide VI-13 by peptidome analysis, which was recognized as the part of glutamyltransferase signal peptide. We modified VI-13 by inserting four arginines and obtained an analog peptide VI-17 to improve its solubility. Our analyses showed that the peptide VI-17 induced rapid context-dependent cell death, and exhibited a higher sensitivity on hepatoma cells, which is attenuated by polyethylene glycol but not necrotic inhibitors such as z-VAD-fmk or necrostatin-1. Morphologically, VI-17 induced cell swelling, blebbing and membrane rupture with release of cellular ATP and LDH into extracellular media, which is hallmark of oncotic process. Mechanistically, VI-17 induced cell membrane pore formation, degradation of α-tubulin via influx of calcium ion. These results indicated that the novel peptide VI-17 induced oncosis in HCC cells, which could serve as a promising lead for development of therapeutic intervention of HCC.

Agrin para-secreted by PDGF-activated human hepatic stellate cells promotes hepatocarcinogenesis in vitro and in vivo.

Evaluating the process and mechanism of fibrogenesis is essential in hepatocellular carcinoma (HCC), especially in hepatocyte transformation and oncogenic signaling. We evaluated the oncogenic role of agrin secreted by platelet-derived growth factor (PDGF)-induced hepatic stellate cell (HSC) in HCC. Cells were co-cultured to investigate the effect of activated HSC on hepatocytes. Liquid chromatography and protein profiling analysis were used to search the distinct proteins secreted in HSC supernatant. Sprague Dawley rats with Diethylnitrosamine (DEN)-induced HCC were used to simulate human liver cancer and sorafenib was administered to investigate its effect on hepatocarcinogenesis. A paired "two-tailed" Student t-test and chi-square tests was used for statistical analysis. PDGF acted as an activator of the HSC and sorafenib inhibits the activation by blocking the combination of PDGF and PDGF receptor. The supernatant of activated HSCs promoted the proliferation, metastasis, and invasion of HL-7702 and SMMC-7721, as well as epithelial-mesenchymal transition (EMT). Agrin found in the HSC supernatant showed the same effect on SMMC-7721 as to the supernatant of activated LX-2. Furthermore, downregulation of agrin by siRNA could decrease the proliferation, metastasis, and invasion of SMMC-7721, and promote MET. Sorafenib prevented DEN-induced hepatocarcinogenesis and could alleviate the liver inflammation and fibrosis. Sorafenib could improve the liver function of Sprague Dawley rats by decreasing the serum levels of ALT and AST. These results demonstrate thatPDGF is an effective activator of HSC and sorafenib could inhibit the activation. In vivo experiment suggested sorafenib could alleviate the hepatocarcinogenesis mediated through agrin secretion and could be potential candidate for treatment of cirrhosis.

Dysbindin promotes progression of pancreatic ductal adenocarcinoma via direct activation of PI3K.

Pancreatic ductal adenocarcinoma (PDAC) represents a biggest challenge in clinic oncology due to its invasiveness and lack of targeted therapeutics. Our recent study showed that schizophrenia susceptibility factor dysbindin exhibited significant higher level in serum of PDAC patients. However, the functional relevance of dysbindin in PDAC is still unclear. Here, we show that dysbindin promotes tumor growth both in vitro and in vivo by accelerating the G1/S phase transition in cell cycle via PI3K/AKT signaling pathway. Mechanistically, dysbindin interacts with PI3K and stimulates the kinase activity of PI3K. Moreover, overexpression of dysbindin in PDAC is correlated with clinicopathological characteristics significantly, such as histological differentiation (P = 0.011) and tumor size (P = 0.007). Kaplan-Meier survival curves show that patients with high dysbindin expression exhibit poorer overall survival, compared to those with low dysbindin expression (P < 0.001). Multivariate analysis reveals that dysbindin is an independent prognostic factor for pancreatic ductal adenocarcinoma (P = 0.001). Thus, our findings reveal that dysbindin is a novel PI3K activator and promotes PDAC progression via stimulation of PI3K/AKT. Dysbindin therefore represents a potential target for prognosis and therapy of PDAC.